Affinity small molecules for the EPO receptor

ABSTRACT

Compounds are provided that complex with the modulating domain of erythropoietin receptor (EPO-R) for use with EPO-R to determine the presence of EPO-R, the ability of other molecules to bind to the modulating domain in competitive assays and to induce a signal by EPO-R into a cell when bound by the subject compounds in a physiological environment. The compounds are characterized by having a six-membered heterocyclic ring comprising at least one nitrogen atom and include substituted triazolopyrimidine, pyridazinone, pyridine and piperidine.

[0001] This application is a continuation of provisional patentapplications, Nos. 60/393,361; 60/393,360 and 60/394,110, all filed onJul. 3, 2002, the entire contents of each of which is incorporatedherein by reference.

STATEMENT TO COMPUTER DISK AND SEQUENCE LISTING

[0002] This application includes a sequence listing of 2 sequences and acomputer disk labeled “Sequence Listing for application entitled“Affinity Small Molecules for the EPO Receptor” by Lennart Olsson andTatjana Naranda” containing files “REC105-SEQLIST.prj” dated “Jul. 2,2003” with 990 bytes, which is the PatentIn project file generated usingPatentIn Version 3.0 software provided by the USPTO, and“REC105-SEQLIST.doc”, dated “Jul. 2, 2003” with 21 kilobytes, which isthe generated sequence listing from the PatentIn project fileREC105-SEQLIST.prj using PatentIn Version 3.0 software, all which areherein incorporated. The information recorded in computer readableformat on the incorporated computer disk labeled “Sequence Listing”containing files “REC105-SEQLIST.prj” and “REC105-SEQLIST.doc” areidentical to the incorporated written sequence listing

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention relates generally to non-peptide molecules havingaffinity for the erythropoietin receptor at a modulating site that isother than the erythropoietin binding site and the use of suchmolecules.

[0005] 2. Background Information

[0006] As the cellular pathways are discovered, there is increasinginsight into the different molecules that are involved in thetransduction of a signal resulting from the binding of a ligand to acellular membrane receptor to the transcription and expression of genesin the host cell genome. The development of erythropoietin (“EPO”) hasallowed for the substantial improvement in the treatment of anemia.Anemia may be a natural phenomenon resulting from a genetic defect ormay be as a result of infection or treatment with radiation orchemotherapy. In each of these instances it is necessary for theviability of the host that the red blood cell count or hematocrit remainsubstantially normal. In many situations, the level of treatment iscompromised by the adverse effect on the red blood cell count, so thatthe desired therapy cannot be administered. The availability of EPO hasprovided a means for restoring the red blood cell count in compromisedpatients.

[0007] EPO is a large glycosylated protein that is expensive anddifficult to manufacture, purify and formulate. It is also degraded byproteases when in the blood, so that a substantial portion of theadministered EPO is lost. Despite these problems, the dramatic effectachieved by EPO has resulted in annual sales exceeding a billiondollars.

[0008] In U.S. Pat. No. 6,333,031 and in Activation of ErythropoietinReceptor Through a Novel Extracellular Binding Site, Naranda, et alEndocrinology 19XX 143(6):2293-2302; Activation of ErythropoietinReceptor in the Absence of Hormone by a Peptide That Binds to a DomainDifferent from the Hormone Binding Site, Naranda, et al., Proc. Natl.Acad. Sci. 1999, 96(13):7569-74 are reported the existence of anextracellular binding site of the EPO receptor (“EPO-R”) referred to asthe “modulation domain.” In the human EPO-R, the modulation domaincorresponds to about amino acids 194-216 and has the amino acid sequenceQRVEILEGRTECVLSNLRGRTRY (SEQ ID NO:1). Binding of a 23 amino acidpeptide having the sequence SEQ ID NO:1 to the EPO-R modulating domainresulted in modulation of the activity of the EPO-R in the presence orabsence of the ligand. The peptide, therefore, offers an auxiliarycompound for use with EPO to enhance the activity of EPO and reduce therequirement for EPO to achieve analogous activity. The peptide can bereadily synthesized efficiently and economically. However, the peptideis small and will be rapidly degraded in the blood. Also, it suffersfrom the inconvenience of requiring injection.

[0009] There is, therefore, an interest in developing small non-peptidemolecules that can serve in conjunction with EPO in the treatment ofanemia, as well as in other functions associated with assays for EPO-R,studying EPO and EPO-R function, purification of EPO-R, and analogoususes.

Relevant Literature

[0010] U.S. Pat. No. 6,333,031 and in Activation of ErythropoietinReceptor Through a Novel Extracellular Binding Site, Naranda, et alEndocrinology 19XX 143(6):2293-2302; Activation of ErythropoietinReceptor in the Absence of Hormone by a Peptide That Binds to a DomainDifferent from the Hormone Binding Site, Naranda, et al., Proc. Natl.Acad. Sci. 1999, 96(13):7569-74, describe the modulation domain and SEQID NO: 1. PCT/IUS02/064211 describes triazolopyrimidines for use asthrombin inhibitors.

SUMMARY OF THE INVENTION

[0011] Organic compounds having an azaaryl heterocyclic core and havingat least 2 substituents are provided that find use as EPO-R modulatingcompounds, competing with SEQ ID NO:1 for the modulating domain site.The compounds can be used to bind to EPO-R for modulating the activityof the EPO-R, isolating EPO-R, evaluating the activity of compoundscapable of upregulating the expression of EPO-R or its endocytosis, aswell as other applications associated with affinity binding. Methods ofpreparation of the compounds are provided. The compounds are provided incombination with a polypeptide comprising the modulating domain ofEPO-R, or instructions for use to bind EPO-R for assays, EPO-Rmodulation and EPO-R purification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows a graphical representation of a competitive bindingassay that may be used to identify non-peptide EPO-R binding molecules.

[0013]FIG. 2 shows a summary of the methods used for assessment ofnon-peptide EPO-R modulators biological activity.

[0014]FIG. 3 shows a graph of the proliferative effect of non-peptideEPO-R modulator E5 in TF-1 cells.

[0015]FIG. 4 shows non-peptide EPO-R modulator E5 activation of EPO-R inUT-7 cells.

[0016]FIG. 5 shows the effect of non-peptide EPO-R modulator E5A24 onerythroid colony formation in methylcellulose. Fetal liver cells wereisolated and seeded in the presence of compound. The colonies werecounted on day 3.

[0017]FIG. 6 shows the effect of non-peptide EPO-R modulator E5 onerythroid colony formation in methylcellulose. Human bone marrow cellswere isolated and seeded in the presence of compound. The colonies werecounted on day 14.

[0018]FIG. 7 shows the cooperation between non-peptide EPO-R modulatorE5 and EPO on erythroid colony formation in methylcellulose. CD34+cellswere isolated and seeded in the presence of compound. The colonies werecounted on day 14.

[0019]FIG. 8 shows cooperation between non-peptide EPO-R modulatorEM5A24 and EPO on erythroid colony formation in methylcellulose. Humanbone marrow cells were isolated and seeded in the presence of compound.The colonies were counted on day 14.

[0020]FIG. 9 shows the effect of non-peptide EPO-R modulator E5 onhematocrit levels in carboplatin-treated 8 week old C57BL mice. Thecompound was given i.p.

[0021]FIG. 10 shows the cooperative effect between non-peptide EPO-Rmodulator E6 and EPO on hematocrit levels in carboplatin-treated 8 weekold C57BL mice. The compound was given i.p.

[0022]FIG. 11 shows the effect of non-peptide EPO-R modulator E6 onhematocrit levels in carboplatin-treated 8 week old C57BL mice. Thecompound was given orally.

[0023]FIG. 12 shows the effect of non-peptide EPO-R modulator E5 onreticulocyte levels in normal mice. The compound was given i.p.

[0024]FIG. 13 shows the effect of non-peptide EPO-R modulators E5A24 andEM5 on up-regulation of Bcl-x_(L) expression in TF-1 cells.

[0025]FIG. 14 shows the effect of non-peptide EPO-R modulators E5A24 andEM5 on up-regulation of Bcl-x_(L) expression in UT-7 cells.

[0026]FIG. 15 shows the effect of non-peptide EPO-R modulators E5A24 andEM5 on increased cell viability of P19 cells after the withdrawal ofserum.

[0027]FIG. 16 shows the effect of non-peptide EPO-R modulators E5A24 andE5A29 on increased cell survival of cortical neurons after glutamatechallenge.

[0028]FIG. 17 shows a summary of activity for non-peptide EPO-Rmodulators.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Organic compounds, having a substituted azaheterocycle of 6annular members with from 1-2 nitrogen atoms in the ring, usually havingat least one site of unsaturation, including azaaryl, as the core, areprovided for binding to EPO-R at what is referred to as the “modulatingdomain.” The compounds have at least two substituents and at least tworings, which may be fused, unfused or both. There will be at least oneheterocyclic ring and up to 4 rings, where an aromatic ring substituentmay be carbocyclic or heterocyclic and of 5-6 annular members. Thesubstituents may be bonded to an annular carbon atom by a carbon atom orheteroatom. The compounds will have at least 12 atoms other thanhydrogen and beside the core may contain aliphatic, aromatic, alicyclic,heterocyclic, and heterosubstituents.

[0030] The azaheterocycle that forms the core of the subject organiccompounds will be substituted with various substituents, whichsubstituents include aliphatic groups including carbocyclic group,aromatic groups and heterocyclic groups, both aromatic and non-aromatic.These in turn may be further substituted with various groups. Among themonovalent groups are included halo, CN, OH, OCH₃, NO₂, SH, NH₂, CF₃,alkyl e.g. CH₃, which in turn may be substituted with the indicatedmonovalent substituents other than alkyl. Divalent substituents willusually be bonded to a ring to form a fused ring, generally having 4carbon atoms and forming a 6-membered carbocyclic ring with the adjacentannular atoms to which the divalent substituent is attached, whereinsuch ring may be further substituted with any of the substituentsindicated above, including a divalent substituent.

[0031] The subject compounds can be provided in combination with apolypeptide comprising the modulating domain, a portion of theextracellular portion of EPO-R, EPO-R in purified form or in conjunctionwith other compounds, as a microsome, lysate, a mixture of polypeptidesand/or organic molecules other than polypeptides. The subject compoundscan be provided as separate from the modulating domain or bound to themodulating domain to form a complex. The compounds are provided incombination with a polypeptide comprising the modulating domain ofEPO-R, or instructions for use to bind EPO-R for assays, EPO-Rmodulation and EPO-R purification.

[0032] The modulating domain is described in the literature as SEQ IDNO:1 for the human EPO-R (“hEPO-R”) and the analogous sequenceQRVEVLEGRTECVLSNLRGGTRY (SEQ ID NO:2) for the mouse EPO-R (“mEPO-R”),where these peptides bind to the modulating domain and affect theactivity of the EPO-R in the presence of EPO binding to the EPO-R. (Thepeptides will hereafter be referred to as “ERP”.) The sequences arereported in the art, Swissprot accession no. P19235 and P14753,respectively. For the most part, the modulating domains from differentspecies will be highly conserved, generally differing by fewer thanabout 5 amino acids. Cross-reactivity is observed between the peptides,where the modulating domain peptide from one species will bind to anEPO-R receptor from a different species.

[0033] In referring to the EPO-R receptor is intended any mammalianEPO-R, particularly primate, more particularly human, rodentiae, murine,hamster and rat, or domestic animals, e.g. bovine, equine, porcine, etc.

[0034] The subject compounds share common features in having aheterocycle core of 6 annular atoms, having at least one nitrogenannular heteroatom and at least two substituents and/or a fused ring.The heterocyclic core includes triazolopyrimidine, particularly[1,2,4]triazolo[1,5-a]pyrimidine, pyridine, pyridazine and[4H,8H]-1,2,8-triazaindene. The compounds will have at least 12 atomsother than hydrogen, usually at least 15 atoms other than hydrogen, andnot more than about 40, usually not more than 36, atoms other thanhydrogen, where the atoms include carbon, chalcogen (O and S), halo,nitrogen and physiologically acceptable metal ions, generally Groups Iand II of the Periodic Chart. Heteroatom containing substituents includeoxy (hydroxyl and ether, where oxygen is bonded to hydrogen or anorganic group) thio (thiol, thioether, sulfinic ester, sulfoxide,sulfone, sulfonic acid, sulfonic acid ester, sulfenamide, sulfonamide,etc., where sulfur is bonded to hydrogen, oxygen, nitrogen or an organicgroup), non-oxo-carbonyl (carboxylic acid, carboxylic acid salt, ester,and amide and the nitrogen, e.g. amidine, amic ester, N-oxy substituted,etc. and thio, e.g. thionocarboxylic acid, thiocarboxylic ester, etc.,analogs thereof), oxo-carbonyl (aldehydo and keto), amino (I°, II°,III°, and IV° derivatives thereof, e.g. dialkylamino, carbamyl,amidinyl, N-oxy amidinyl, ureido, tetraalkylamino, etc.), halo(fluorine, chlorine, bromine, iodine and their anions), and the like.Groups substituted on the core heterocycle through a carbon atom includealiphatic, aliphatically saturated and unsaturated, generally having notmore than 3 sites, usually not more than 2 sites, of unsaturation,alicyclic (included within aliphatic unless separately defined),carbocyclic aromatic of from 6-12, usually 6-10, annular members andheterocyclic of from 5 to 12, usually 5 to 10, annular members, andhaving from 1 to 4, usually 1 to 3 heteroannular members, usuallychalcogen and nitrogen, where each of these rings may have from 0 to 4,usually 0 to 2 heteroatom containing substituents. The total number ofcarbon atoms will be in the range of about 9 to 20, usually 9 to 18,carbon atoms, while the number of heteroatoms will generally be in therange of about 3 to 12, more usually 4 to 10.

[0035] The four main core structures have the following formulas:

[0036] (1) triazolopyrimidines

[0037] wherein the groups present are defined as follows:

[0038] X is a heteroatom linking group of from 1 to 7, usually 1 to 3atoms other than hydrogen, including amino and substituted amino havingan alkyl group of from 1 to 6, usually 1 to 2 carbon atoms, and usuallyunsubstituted, thio, sulfoxide and sulfone (sulfur having from 0-2oxygen atoms), or oxy;

[0039] n is 0 or 1;

[0040] is hydrogen or an organic group of from 1 to 12, usually 1 to 10carbon atoms and from 0 to 6, usually 0 to 4 heteroatoms, which arechalcogen, nitrogen, and halogen where the organic group may bealiphatic having from 0 to 4, usually 0 to 2 sites of unsaturation, e.g.alkyl, alkenyl and alkynyl, including alicyclic having from 0 to 2 sitesof unsaturation, and includes alkyl of from 1 to 6 carbon atoms, usually1 to 3, more usually 1 to 2, carbon atoms, non-oxo-carbonyl and thenitrogen and sulfur derivatives thereof, particularly the nitrogenderivatives thereof, of from 3 to 12 atoms other than hydrogen,particularly 1 to 10 carbon atoms and 2 to 6, usually 2 to 4heteroatoms, aryl, particularly phenyl, heterocyclic of from 3 to 6annular members, and combinations thereof, e.g. aralkyl and alkaryl,where the cyclic structures may have from 1 to 3, commonly 1 to 2 rings,which may be fused or unfused, illustrated by methyl, ethyl, propyl,hexyl, phenyl, tolyl, fluorophenyl, anisole, phenetole, naphthyl,indolyl, quinolinyl, carboxy, ethoxycarbonyl, amidinyl,hydroxyamidinylmethyl, ethoxycarbamylethyl, phenylthioethyl,phenylaminopropyl, nitrophenethyl, p-nitrophenyl, mesitylethyl,2-fluoro-5-ethylphenyl, 2,6-difluorophenyl, including the partial orcompletely reduced forms of the aromatic groups, e.g. pyridine andpiperidine, etc., more particularly having a 6 membered aromatic group,particularly phenyl, having from 0 to 4, usually 0 to 3 substituents,which include alkyl of from 1 to 3 carbon atoms, particularly methyl,halo, particularly Cl and F, oxy of from 0 to 3 carbon atoms,trihalomethyl, cyano and nitro. R₁ may have an aryl group of from 6 to10 carbon atoms, particularly phenyl, when X is nitrogen and n is 1.

[0041] R₂ is hydrogen, a heterofunctionality having nitrogen and/orchalcogen, which heterofunctionality is bonded to an annular carbonatom, where the heteroatom may be substituted with an organic group offrom 1 to 10, usually 1 to 8 carbon atoms, and from 0 to 4, usually 0 to3 heteroatoms, particularly alkyl of from 1 to 6, usually 1 to 2, carbonatoms, aryl, including alkaryl, aralkyl and aralkenyl of from 5 to 10carbon atoms, aroyl of from 6 to 10 carbon atoms, or an organic groupbonded through a carbon atom to the annular atom and of from 1 to 12,usually 1 to 8 carbon atoms, including aliphatic, alicyclic, aromaticand heterocyclic groups, having from 0 to 4, usually 1 to 2 heteroatoms,as described above for R₁;

[0042] R₃ is hydrogen or an organic group of from 1 to 10, usually 1 to8 carbon atoms and from 0 to 4, usually 0 to 2 heteroatoms, usuallychalcogen and nitrogen, particularly oxygen, including alkyl,oxycarbonyl, particularly alkoxycarbonyl, and aryl, alkaryl and aralkyl,particularly carbocyclic aryl, particularly of from 6 to 8 carbon atoms;and

[0043] R₄ is hydrogen or alkyl and substituted alkyl of from 1 to 6,usually 1 to 3 carbon atoms, where the substituents are oxy, amino andhalo, particularly fluoro; and

[0044] where R₃ and R₄ may be taken together to form a ring with theatoms to which they are attached, having from 4 to 10, usually 4 to 8annular atoms, which may be substituted or unsubstituted, particularlycomprising a 1,4-butandiyl group having from 0 to 1 site of unsaturationto form a fused ring and adding from 1 to 2 rings, where the butandiylmay be fused to an additional ring so as to be a bicyclo substituent,wherein the annular atoms may be substituted with chalcogen, nitrogen,halo and alkyl of from 1 to 3 carbon atoms, particularly oxy of from 0to 3 carbon atoms, thio of from 0 to 3 carbon atoms and amino of from 0to 4 carbon atoms;

[0045] (2) di(arylthio)pyridines

[0046] wherein:

[0047] p is 0, 1 or 2, usually 2; and

[0048] R₅ is a group having from 1 to 3 atoms other than hydrogen andincludes oxy, thio, amino, nitro, cyano, and alkyl, wherein R₅ is at the3 or 4 position, usually the 3 position;

[0049] (3) aryl substituted pyridazinone (includes dihydropyridazine):

[0050] wherein:

[0051] Y is O, S(O)_(m),, wherein m is 0, 1 or 2, amino or CH₂;

[0052] R₆ is H or alkyl of from 1-3 carbon atoms;

[0053] R₇ is hydrogen, or a group of from 0 to 3 atoms other thanhydrogen, and includes oxy, thio amino, nitro, cyano, and alkyl;

[0054] V is an aryl group having 6 annular members comprising 0 to 2nitrogen atoms and the remainder carbon atoms

[0055] U is a substituent group of from 0 to 5 atoms other thanhydrogen, and includes oxy, thio amino, nitro, cyano, halo, and alkyl,usually of from 1 to 3 carbon atoms, while u is 0 to 3, usually 0 to 1;

[0056] (4) diazolohexahydroquinoline

[0057] wherein:

[0058] Y is oxygen, sulfur, NH, (alkyl of from 1 to 3 carbon atoms, H)or H₂;

[0059] R₇ is hydrogen, a heterosubstituent of from 1 to 3 heteroatoms asdescribed previously, or an organic group of from 1 to 12, usually 1 to10, more usually 1 to 6 carbon atoms and from 0 to 6, usually 0 to 4heteroatoms, which are chalcogen, nitrogen, and halogen where theorganic group may be aliphatic having from 0 to 4. usually 0 to 2 sitesof unsaturation, e.g. alkyl, alkenyl and alkynyl, including alicyclichaving from 0 to 2 sites of unsaturation, and includes alkyl of from 1to 6 carbon atoms, usually 1 to 3, more usually 1 to 2, carbon atoms,non-oxo-carbonyl and the nitrogen and sulfur derivatives thereof,particularly the nitrogen derivatives thereof, of from 3 to 12 atomsother than hydrogen, particularly 1 to 10 carbon atoms and 2 to 6,usually 2 to 4 heteroatoms, where the aliphatic groups may besubstituted with from 1 to 4, usually 1 to 2 heterosusbtituents, whichhave been described above, aryl, particularly phenyl, heterocyclic offrom 3 to 6 annular members, and combinations thereof, e.g. aralkyl andalkaryl, where the cyclic structures may have from 1 to 3, commonly 1 to2 rings, which may be fused or unfused, illustrated by methyl, ethyl,propyl, hexyl, phenyl, tolyl, fluorophenyl, anisole, phenetole,naphthyl, furanyl, thienyl, indolyl, quinolinyl, carboxy,ethoxycarbonyl, amidinyl, hydroxyamidinylmethyl, ethoxycarbamylethyl,phenylthioethyl, phenylaminopropyl, nitrophenethyl, p-nitrophenyl,mesitylethyl, 2-fluoro-5-ethylphenyl, 2,6-difluorophenyl, including thepartial or completely reduced forms of the aromatic groups, e.g.pyridine and piperidine, etc., more particularly having a 6 memberedaromatic group, particularly phenyl, having from 0 to 4, usually 0 to 3substitutents, which include alkyl of from 1 to 3 carbon atoms,particularly methyl, halo, particularly Cl and F, oxy of from 0 to 3carbon atoms, trihalomethyl, cyano and nitro.

[0060] R₈ is hydrogen, a heterosubstituent of from 1 to 3 heteroatoms asdescribed above, or an organic group of from 1 to 12, usually 1 to 10carbon atoms, more usually 1 to 6 carbon atoms, and from 0 to 6, usually0 to 4 heteroatoms, which are chalcogen, nitrogen, and halogen where theorganic group may be aliphatic having from 0 to 4, usually 0 to 2 sitesof unsaturation, e.g. alkyl, alkenyl and alkynyl, including alicyclichaving from 0 to 2 sites of unsaturation, and includes alkyl of from 1to 6 carbon atoms, usually 1 to 3, more usually 1 to 2, carbon atoms,non-oxo-carbonyl and the nitrogen and sulfur derivatives thereof,particularly the nitrogen derivatives thereof, of from 3 to 12 atomsother than hydrogen, particularly 1 to 10 carbon atoms and 2 to 6,usually 2 to 4 heteroatoms, aryl, particularly phenyl, heterocyclic offrom 3 to 6 annular members, and combinations thereof, e.g. aralkyl andalkaryl, where the cyclic structures may have from 1 to 3, commonly 1 to2 rings, which may be fused or unfused, or a heterosusbtituent of from 1to 3 heteroatoms as described previously, illustrated by methyl, ethyl,propyl, hexyl, phenyl, tolyl, fluorophenyl, anisole, phenetole,naphthyl, indolyl, quinolinyl, carboxy, ethoxycarbonyl, amidinyl,hydroxyamidinylmethyl, ethoxycarbamylethyl, phenylthioethyl,phenylaminopropyl, nitrophenethyl, p-nitrophenyl, mesitylethyl,2-fluoro-5-ethylphenyl, 2,6-difluorophenyl, including the partial orcompletely reduced forms of the aromatic groups, e.g. pyridine andpiperidine, etc., more particularly having a 6 membered aromatic group,particularly phenyl, having from 0 to 4, usually 0 to 3 substitutents,which include alkyl of from 1 to 3 carbon atoms, particularly methyl,halo, particularly Cl and F, oxy of from 0 to 3 carbon atoms,trihalomethyl, cyano and nitro. More particularly, R₈ is hydrogen, alkylof from 1 to 6, usually 1 to 3 carbon atoms or substituted alkyl, wherethe substituents have been described in relation to or a heterocycle offrom 5 to 6 annular members and from 1 to 2 heteroannular members thatare oxygen, nitrogen or sulfur, having a total number of atoms otherthan hydrogen in the range of 5 to 10, usually 5 to 8; and

[0061] R₉, R₁₀, R₁₃, R₁₄, R₁₅ and R₁₆ are the same or different, usuallynot more than two, more usually not more than one, being other than H;when not H, the groups will come within the definition of R₈, generallybeing alkyl or substituted alkyl of from 1 to 6, usually 1 to 3 carbonatoms, having 0 to 2, usually 0 to 1 substituent as described above, aheterosubstituent of from 1 to 3 heteroatoms that are chalcogen,nitrogen or halo, oxy, thio or amino of from 0 to 8, usually 0 to 6carbon atoms, wherein aliphatic, aromatic and heterocyclic substituentsmay have in turn from 0 to 2 substituents as described above,particularly alkyl or substituted alkyl of from 1 to 4, usually 1 to 3carbon atoms

[0062] R₁₁ and R₁₂ are the same or different and are H or an organicgroup of from 1 to 12, usually 1 to 8, more usually 1 to 6 carbon atomsand 0 to 6, usually 0 to 4 heteroatoms that are chalcogen, nitrogen andhalo, particularly aliphatic (including alicyclic) of from 1 to 8,usually 1 to 3 carbon atoms, substituted or unsubstituted, having from 0to 3, usually 0 to 2, substituents as described above, aromatic of from6 to 12, usually 6 to 10 carbon atoms, substituted or unsubstituted,having from 0 to 3, usually 0 to 2, substituents as described above, andheterocyclic having from 3 to 6 annular members with from 1 to 3heteroatoms that are chalcogen and nitrogen; particularly hydrogen oralkyl of from 1 to 3 carbon atoms.

[0063] Of the four formulas, the core structure of thetriazolopyrimidine is of particular interest. This family of compoundsis recognized by having at the 2-position ((X)_(n)—R₁), where X isamino, thio or sulfone, n is 0 or 1, where the amino may beunsubstituted or substituted with lower alkyl of from 1 to 3 carbonatoms, and R₁ is a lower alkyl group of 1 to 3 carbon atoms,particularly methyl, or an organic group having a six annular memberedaromatic group, particularly phenyl or pyridinyl group, preferablyphenyl, having from 0 to 3 substituents, where the substituents arehalo, particularly chloro, lower alkyl of from 1 to 3 carbon atoms,particularly methyl, nitro, trihalomethyl, particularly trifluoromethyl,and where the phenyl group is terminal and is either directly bonded tothe amino or thio atom or bonded through a linking group of from 1 to 4,usually 1 to 3, atoms in the chain, being carbon, nitrogen, orchalcogen, particularly carbon and nitrogen, where heteroatoms arebonded solely to carbon and hydrogen, there being from 0 to 2heteroatoms in the chain, the linking group generally being a total offrom 1 to 8 atoms, usually 1 to 6 atoms other than hydrogen and havingfrom 0 to 2 heterosusbtituents, e.g. oxo, or having alpha-acetamidinylhaving from 0 to 1 N—OH, i.e. N-hydroxy alpha-acetamidinyl.

[0064] R₂ is a carbocyclic bonded to the annular carbon atom by alinking group of from 1 to 3 atoms having from 0 to 1 heteroatom that ischalcogen or nitrogen, particularly amino (—NH—), wherein the ring hasfrom 3 to 6 annular atoms, particularly 3 to 4, a group of from 0 to 3carbon atoms that is lower alkyl of from 1 to 3 carbon atoms,particularly methyl, oxy, or amino, phenyl or substituted phenyl bondeddirectly to an annular atom or through a carbamyl or vinyl group, wherethe substituents are oxy of from 0 to 3 carbon atoms, particularlyalkoxy, more particularly methoxy, or lower alkyl of from 1 to 3 carbonatoms, particularly methyl, or a heterocycle of from 5 to 6 annularmembers, having from 1 to 2, usually 1, annular heteroatom, where theannular heteroatoms are chalcogen and nitrogen, and having from 0 to 1substituent of from 0 to 3 carbon atoms. Illustrative groups includeamino, ethylamino, dimethylamino, hydroxy, propyloxy, ethoxy, methoxy,phenyl, tolyl, mesityl, phenetole, anisole, chlorophenyl, bromotolyl,styryl, p-ethoxystyryl, phenethylamine, benzylamine, N-phenylaminocarbonyl (—NHCO—), N-2,4-dichlorophenyl aminocarbonyl, N-anisolylaminocarbonyl, pyridinylmethyl, thiophenyl, thiazolinyl and piperidinyl,as well as the groups described for R₃.

[0065] R₃ is hydrogen or an organic group of from 1 to 8 carbon atomsand 0 to 4, usually 0 to 3 heteroatoms, the heteroatoms being chalcogen,nitrogen and halo, being aliphatic, unsaturated or saturated,particularly saturated, including alkyl of from 1 to 6 carbon atoms andhaving 0 to 2, usually 0 to 1, site of aliphatic unsaturation, non-oxocarbonyl of from 1 to 4 carbon atoms, carbocyclic bonded to the annularcarbon atom by a linking group of from 1 to 3 atoms having from 0 to 1heteroatom that is chalcogen or nitrogen, particularly amino (—NH—),wherein the ring has from 3 to 6 annular atoms, particularly 3 to 4, andalkoxycarbonyl, where the alkoxy group is of from 1 to 3 carbon atoms.Illustrative groups include methyl, propyl, allyl, propargyl,3-butenyl-1, cyclopropylethyl, cyclobutylmethyl, cyclopropylpropylamino,cyclopropylmethoxy, cyclopentyl, allylamino, hexoxy, butoxyformyl,phenyloxyformyl, and methoxyethylformyl.

[0066] R₄ is hydrogen, lower alkyl of from 1 to 3 carbon atoms,particularly methyl, alkoxyalkyl of from 2 to 5, usually 2 to 4 carbonatoms, or haloalkyl of from 1 to 3 carbon atoms and from 1 to 4 haloatoms, usually fluoro, more particularly trifluoromethyl, where R₄ ispreferably hydrogen or lower alkyl of from 1 to 3 carbon atoms,particularly methyl. Illustrative groups include methyl, propyl,isobutyl, 4-methylpentyl, 3-chloropropyl, 4,4,4-trifluorobutyl,2-fluoroethyl, trifluoromethyl, and 2,3-difluoropropyl.

[0067] In referring to aliphatic groups, the aliphatic groups willinclude alkyl groups that are branched or straight chain, generallyhaving from 1 to 6, usually 1 to 3 carbon atoms, and having from 0 to 3heteroatoms that are chalcogen, nitrogen and halogen, usually being oxyof from 0 to 3 carbon atoms, amino of from 0 to 4 carbon atoms, and thiohaving from 0 to 2 oxygen atoms and of from 0 to 3 carbon atoms, beingsaturated or unsaturated having from 0 to 3 sites of unsaturation,namely double and triple bonds.

[0068] In referring to aryl groups, both carbocyclic and heterocyclicgroups are intended, particularly nitrogen heterocycles, where therewill be from 5 to 12, usually 5 to 10, annular atoms, more particularly5 or 6 annular atoms, and having from 0 to 8, usually 0 to 6, morefrequently 0 to 3 heteroatoms, as chalcogen, nitrogen, and halo, andbeing substituted or unsubstituted, where the substituents will be offrom 1 to 8, usually 1 to 6 atoms other than hydrogen, generally havingfrom 0 to 6, usually 0 to 3 carbon atoms, and include oxy, thio, amino,non-oxo-carbonyl, including the nitrogen and sulfur analogs thereof,oxo-carbonyl, halo, cyano, nitro, etc..

[0069] In referring to heterocyclic groups, these groups will begenerally non-aromatic and have from 5 to 10, usually 5 or 6 annularmembers, having from 1 to 3, usually 1 to 2, heteroatoms per ring, whichare chalcogen and nitrogen, being saturated or unsaturated, generallyhaving from 0 to 3, usually 0 to 2 sites, of ethylenic unsaturation, andbeing unsubstituted or substituted, having from 0 to 3 substituents.

[0070] Substituents will generally include alkyl of from 1 to 6, usually1 to 3, carbon atoms, haloalkyl of from 1 to 3 carbon atoms and from 1to 7, usually 1 to 5, halo atoms, particularly fluoro, halo,particularly Cl, oxy of from 0 to 3 carbon atoms, thio of from 0 to 8,particularly 6 to 8, carbon atoms having 0 to 2 oxygen atoms bonded tosulfur, amino of from 0 to 8 carbon atoms, usually when alkylamino offrom 1 to 4 carbon atoms, when aryl amino of from 6 to 8 carbon atomsand when aroylamino of from 7 to 8 carbon atoms, nitro, cyano, halo,particularly fluorine and chlorine, and non-oxo-carbonyl of from 1 to 6,usually 1 to 3, carbon atoms, primarily acid and ester.

[0071] Of particular interest are the triazolopyrimidines. Moreparticularly, are those compounds that modulate the activity of theEPO-R, where modulation intends that a detectable signal is transducedinto the cell upon binding of the compound, e.g. expression of aprotein. Compounds coming within this class are indicated using thedesignation of the above formula are as follows. These compounds willusually have X as S or NH. R₁ will usually be alkyl of from 1 to 3carbon atoms, particularly CH₃, substituted phenyl bonded directly to anannular carbon atom or through a linking group of from 1 to 3 atoms inthe chain having from 0 to 3, usually 1 to 2, the atoms being carbon andnitrogen, more particularly methylene and aminoethylene, and the phenylgroup being unsubstituted or having substituents that are CH₃, Cl, NO₂,and CF₃,. R₂ is CH₃, NH₂, OH, and aroylamido of from 7 to 8 carbon atomshaving from 0 to 2 susbtituents that are CH₃, Cl, NO₂, and CF₃,particularly toluoylamido. R₃ is cycloalkylalkyl of from 4 to 8, usually4 to 6 carbon atoms, having from 3 to 4 annular atoms, H or carboxy. R₄is H, lower alkyl of from 1 to 3 carbon atoms, particularly CH₃, oralkoxymethyl of from 2 to 4 carbon atoms, particularly methoxymethyl. R₃and R₄ may be taken together to definephenyl-1,2-dimethylene-alpha-halo, alpha-CH₃, where the rings may befurther susbtituted with from 1-2 substituents that are halo, e.g. F andCl, NO₂, CH₃, and CF₃.

[0072] When R₃ is an aliphatic group (aliphatic intends alkyl,cycloalkyl, and combinations thereof, whether aliphatically saturated orunsaturated), or an aliphatic group substituted amine of from 2 to 6,usually 3 to 5 carbon atoms, wherein cycloalkyl is of from 3 to 4 carbonatoms, generally saturated, X, n, R₁, R₂, and R_(4,) will have thefollowing definitions. X is NH or S and n is 1. R₁ is alkyl of from 1 to3 carbon atoms, particularly methyl, or phenyl having from 0 to 3substitutents, particularly 2 to 3 substituents that include CH₃, CF₃,NO₂, and Cl, linked to X directly or through a linking chain of from 2to 4 atoms in the chain consisting of C and N, wherein N is bondedsolely to carbon, and having from 0 to 1 non-oxo carbonyl group; R₂ isH, alkyl of from 1 to 3 carbon atoms, particularly CH₃, OH, OCH₃, andNH₂; and R₄ is H and CH₃. When R₂ has the aliphatic group, then thedefinition of R₃ will be substituted by the definition of R₂, with theother definitions remaining the same.

[0073] When R₃ is H, X, n, R₁, R₂ and R₄ will have the followingdefinitions. X is NH or S and n is 1. R₁ is alkyl of from 1 to 3 carbonatoms, particularly methyl, or phenyl having from 0 to 3 substitutents,particularly 2 to 3 substituents that include CH₃, CF₃, NO₂, and Cl,with the phenyl group linked to X directly or through a linking chain offrom 1 to 4 atoms in the chain consisting of C and N, wherein N isbonded solely to carbon, and having from 0 to 1 non-oxocarbonyl group,or amidinyl methyl having from 0 to 1 N-hydroxyl group; R₂ is H, alkylof from 1 to 3 carbon atoms, particularly CH₃, OH, OCH₃ and NH₂, aheterocycle having 5 to 6 annular members, particularly 5, where theheteroatoms are chalcogen and nitrogen, particularly chalcogen andhaving from 0 to 2, usually 1 to 2, sites of unsaturation, or phenylhaving from 0 to 2, usually 1-2 substituents, that are CH₃, Cl, OCH₃,and NO₂, where phenyl is bonded to the annular carbon atoms directly orthrough an aliphatic hydrocarbon chain of from 1 to 3, usually 1 to 2carbon atoms, having from 0 to 1 site of ethylenic unsaturation; and R₄is H and CH₃.

[0074] When R₃ comprises a non-oxo-carbonyl group (particularly acid,ester or amide) of from 1 to 5, usually 1 to 4, carbon atoms,particularly 1 or 3), wherein the oxo group is bonded directly to anannular atom, X, n, R₁, R₂ and R₄ will have the following definitions. Xis NH or S and n is 1. R₁ is alkyl of from 1 to 3 carbon atoms,particularly CH₃, or phenyl having from 0 to 2 substituents that areCH₃, Cl, NO₂ or OCH₃, particularly methyl and phenyl is bonded directlyto an annular atom or through a linking group of from 1 to 3 atoms inthe chain, particularly carbon and nitrogen, usually CH₂; R₂ is CH₃, OH,OCH₃, or NH₂, particularly OH or NH₂; and R₄ is H or CH₃, particularlyH.

[0075] When R₃ is phenyl having from 0 to 2 substituents and bondeddirectly to an annular atom or through a linking group of from 1 to 3atoms in the chain which are carbon and nitrogen, particularly CH₂,where the substituents are CH₃, Cl, NO₂ and OCH₃ X, n, R₁, R₂ and R₄will have the following definitions. X is NH or S and n is 1. R₁ isphenyl having from 0 to 3 substituents, usually 1 to 2 substituents thatare CH₃, Cl, NO₂ and OCH₃, bonded directly or through CH₂ to an annularatom. R₂ is CH₃, OH, OCH₃, and NH₂, particularly OH and OCH₃. R₄ is H orCH₃.

[0076] Exemplary compounds that have been shown to be active inmodulating EPO-R response to EPO are as follows:

[0077] The following compound has been designated as E5

[0078] The following compound is designated as EM5:

[0079] The triazolopyrimidines of the subject invention are referred toas the E5 (including EM5) family for convenience. The following tablesets forth compounds that have been prepared and tested for binding tothe modulating domain. All of the compounds set forth have specificaffinity to the modulating domain with members of the group providingmodulation of the EPO-R activity in transducing a signal into a cell.

[0080] Table 1 TABLE 1 E5 Analog Non-peptide EPO-R Modulators Cmpnd—(X)_(n)— R₁ R₂ R₃ R₄ E5* NH

H

CH₃ E5-A20 S

CH₃ H CH₃ E5-A24 S

CH₃ H CH₃ E5-A25 S

CH₃ H CH₃ E5-A14 S

CH₃ H CH₃ E5-A2 S CH₃ NH₂ —COOH H E5-A9 NH

OH

E5-A16 S

CH₃ H CH₃ E5-A18 S

CH₃ H CH₃ E5-A4 S CH₃

H H E5-A10 NH

OH H —CH₂—O—CH₃

[0081] Table 2 TABLE 2 E5 Analog Non-peptide EPO-R Non-modulatingBinding Molecules Cmpnd —(X)_(n)— R₁ R₂ R₃ R₄ E5-A1 S CH₃ NH₂

H E5-A3 S CH₃ NH₂ H H E5-A5 S CH₃ OCH₃ H CH₃ E5-A6 —SO₂— CH₃ OCH₃ H CH₃E5-A7 n = 0 H OH H CH₃ E5-A8 S CH₃ OH H CH₃ E5-A11 NH

OH

CH₃ E5-A12 S H CH₃ H CH₃ E5-A13 S CH₃ CH₃ H CH₃ E5-A15 S

OH H CH₃ E5-A17 S

CH₃ H CH₃ E5-A19 S

CH₃ H CH₃ E5-A21 n = 0 H

H

E5-A22 S

OH

H E5-A23 S

OH

H E5-A28 NH H

H H E5-A30 NH H

H H E5-A31 NH H

H H

[0082] For the aryl substituted pyridazinones, the following arepreferred.

[0083] X is thio, particulary sulfone, y is 0 to 1, and

[0084] R₇ is H, halo, particularly Cl, alkyl of from 1 to 3 carbonatoms, particularly methyl, OH or OCH₃.

[0085] The following compound is designated as E6.

[0086] The following compound is designated as E4.

[0087] For the diazolohexahydroquinolines, the following are preferred:

[0088] R₇, R₉₋₁₀ and R₁₃₋₁₆ are the same or different, at least 2 andnot more than 4 being other than H, and are otherwise alkyl orsubstituted alkyl of from 1 to 6, usually 1 to 3 carbon atoms and from 0to 1 substituent, particularly chalcogen and amino, where the heteroatommay be bonded to the annular atom, heterocyclic of from 3 to 6 annularatoms, having from 1 to 2 annular heteroatoms that are chalcogen ornitrogen, and aromatic of from 6 to 8 carbon atoms having from 0 to 2substituents including alkyl, oxy and halo;

[0089] R₈ is H, an organic group of from 1 to 12, usually 1 to 6 carbonatoms or a heterosubstituent of 1 to 3 heteroatoms as described above,where the organic group may be bonded directly to the annular carbonatom or through a heteroatom that is chalcogen or nitrogen, the organicgroup can be aliphatic (including alicyclic) of from 1 to 12, usually 1to 6, more usually 1 to 3 carbon atoms, which group may be substitutedwith substituents as indicated previously, aromatic of from 6 to 10,usually 6 to 8 carbon atoms, or heterocyclic of from 3 to 6 annularmembers, particularly 5 to 6 annular members having from 1 to 3, usually1 to 2 heteroatoms that are chalcogen and nitrogen and may be saturatedor unsaturated, and combinations thereof. Exemplary groups includemethyl, methoxyethyl, cyanoethyl, acetyl, N-methyl carbamyl,trifluoromethyl, cyclopropylmethyl, chlorophenyl, phenetole, pyranyl,furanyl, thiophenyl, pyrrolyl, piperidinyl, pyridinyl, methylpyridinyl,etc.

[0090] R₁₁, and R₁₂ may be the same or different and include H, anorganic group of from 1 to 12, usually 1 to 6 carbon atoms, particularlyaliphatic (including alicyclic), particularly alkyl or acyl, which groupmay be substituted with substituents as indicated previously, aromaticof from 6 to 10, usually 6 to 8 carbon atoms, or heterocyclic of from 3to 6 annular members, particularly 5 to 6 annular members having from 1to 3, usually 1 to 2 heteroatoms that are chalcogen and nitrogen and maybe saturated or unsaturated, and combinations thereof. Illustrativesubstituents include methyl, ethyl, hydroxyethyl, glycolyl, acetyl,butyryl, cyclobutylmethyl, chlorophenyl, tolyl, pyranyl, furanyl,pyrrolyl, pyrrolidinyl, piperidinyl, pyridinyl, pyrazinyl, etc.

[0091] The following compound is of particular interest and isdesignated E5A29

[0092] The subject compounds may be provided in a variety of ways,particularly in combination with other materials, both active andinactive for the purpose intended for the subject compounds. The subjectcompounds may be provided as the only active ingredient or may beprovided as a complex with at least the modulation domain of the EPO-R.In the former situation, the subject compounds will usually be providedwith a variety of diluents to allow for use at low concentrations, e.g.drug formulation or assay reagent. Alternatively, the subject compoundsmay be prepared as complexes with at least the modulating domain of theEPO-R so as to be used in assays for competitive binding, where releaseof the subject compound is to be determined. The subject compound may beprovided together with at least the modulating domain of the EPO-R inseparate containers or in a single container as a dry or liquidformulation, complexed or uncomplexed or both.

[0093] The subject compounds find many applications associated with theEPO-R and EPO. The subject compounds can be used in affinity columnswhere one or more of the subject compounds may be functionalized andbonded to packings for affinity columns. Packings for affinity columnsinclude Agarose, Sepharose, Sephadex, Latex, etc. Numerous literaturereferences describe methods of functionalizing compounds and linkingthem to active or activated particles. Also, one may functionalize gelsfor electrophoresis, where the EPO-R will complex with the subjectcompounds with a reduction in its migratory rate. See, for example, U.S.Pat. Nos. 5,445, 958; 6,033,574; 6,258,275; 6,281,006, and referencescited therein.

[0094] The subject compounds may also be used to count the number ofEPO-R present. Particularly, one may evaluate the effect of changes inthe environment of cells on the up or down regulation of EPO-R asobserved with the EPO-R population at the cellular membrane. By havingthe subject compounds labeled with a detectable label, e.g.radioisotope, fluorescer, etc., after contacting the cells with thelabeled derivative of the subject compounds, one washes awaynon-specifically bound labeled derivative and then measures the amountof labeled derivative still bound to the cells. By employing a standard,where one knows the EPO-R population in a predetermined environment, onecan determine whether the change in environment affects the surfaceEPO-R population. Changes in environment may be test compounds for theability to modulate the EPO-R population by binding to the ligand siteor modulating the expression of EPO-R. The subject compounds may also beused to isolate cells expressing the EPO-R receptor. By binding thesubject compounds to a surface, e.g. container wall, beads,particularly, magnetic beads, capillary wall, etc., one can dispersecells in an appropriate medium and contact the dispersed cells with thebound subject compounds. After washing away non-specifically boundcells, one can isolate cells expressing EPO-R and transporting the EPO-Rto the cell membrane.

[0095] The subject compounds can be used for competitive assays fordetermining whether other compounds bind to the modulating domain. Byusing labeled derivatives of the subject compound to compete with testcompounds, one can evaluate the binding affinity of the test compound.By combining a labeled subject compound and a test compound with EPO-Runder complex forming conditions, the amount of labeled subject compoundbound to the EPO-R will be related to the affinity of the test compound.Various assay protocols are known for competitive assays, for example,immobilizing the EPO-R and measuring the amount of the labeledderivative that binds to the immobilized EPO-R in the presence andabsence of the test compound. With a fluorescer labeled subjectcompound, one may use total internal reflection to detect fluoresceradjacent the wall of the container. Alternatively, one may have abiotinylated subject compound. After combining the biotinylated subjectcompound, the test compound and EPO-R under complex forming conditions,one may separate the complex of the biotinylated subject compound andEPO-R using immobilized strept/avidin. After washing to remove anynon-specifically bound EPO-R, one can add an enzyme labeled anti-(EPO-R)and measure the turnover rate of substrate of the enzyme. The turnoverrate will be related to the affinity of the test compound for themodulating domain. Another method is to use the labeled subject compoundas a complex with at least the modulating domain of the EPO-R andimmobilize the complex. One can then determine the rate at which thelabeled subject compound is released from the complex in the presenceand absence of a test compound, indicating the affinity of the testcompound.

[0096] Various labels find use in assays, such as radioisotopes, e.g.tritium, fluorescers, chemiluminescers, enzymes, quantum dots, etc.Alternatively, one may use uncommon isotopes and detect the compound bymass spectroscopy. The manner in which these various labels areincorporated into the subject compounds is well known by analogy. Forexample, methyl groups may be functionalized with a thiol group, wherebya maleimide-functionalized fluorescer can be attached through athioether group. The acid group of an ester or amide may be replacedwith an acid group of a chemiluminescer. Enzyme functionalizedantibodies can be employed, where the antibody binds to the EPO-Rcomplex with the subject compound.

[0097] The subject compounds form complexes with the extracellularportion of EPO-R. The complex is readily formed by bringing together asubject compound and at least the modulating domain portion of EPO-Runder buffered conditions, having a pH usually in the range of about 5to 10, more usually 6 to 9, including physiological conditions. Variousbuffers may be employed, e.g. tris, phosphate, borate, carbonate, MOPS,etc., generally at a concentration in the range of about 10 to 200 mM.The concentrations of the two components will depend upon thecircumstances under which they are brought together, e.g. a syntheticmixture in an aqueous buffered medium, cells in a culture mediumexpressing EPO-R, administration to a host, etc. The concentration mayrange from 0.1 nM to saturation or greater. The EPO-R may be a purifiedmolecule, a member of a lysate, a member of a mixture of proteins,present in a microsome, or present in a cell membrane, where the cellmay be erythrocytic or other cell, may be a cell transformed with anEPO-R expression construct for expression of EPO-R under inducible orconstitutive conditions, or the like. The complex may be a purifiedcomplex or may be present in a mixture as described above. The EPO-R mayor may not have a ligand, e.g. EPO or mimetic, present in the ligandbinding site of the complex.

[0098] Compounds that have physiological effect can be used inconjunction with a ligand for EPO-R, e.g. erythropoietin. In thepresence of EPO, the non-peptide EPO-R modulators have an effect withEPO in activating EPO-R and transducing a signal into the cell, whereless EPO can be used to obtain the same cellular response in thepresence of a subject compound. For example, when added individually,very low concentrations of EPO and non-peptide EPO-R modulator give noor a very low number of CFU-e from bone marrow; whereas, when addedtogether they give a maximum response.

[0099] The non-peptide EPO-R modulator can be administered to a subject(e.g., mammal) in vivo or to mammalian cells ex vivo or in vitro.

[0100] Enhancement of the cellular response to EPO by the non-peptideEPO-R modulators provides a means of improving the response of subjectsthat are either partially unresponsive, e.g. resistant, to the action ofEPO or have low natural EPO levels. This is particularly desirable insome situations where the administration of EPO may have undesirableside effects or the host has diminished amounts of EPO.

[0101] In one method of use, non-peptide EPO-R modulators may beadministered to subjects requiring enhancement of the response tonaturally occurring levels of EPO. In an alternative method, thenon-peptide EPO-R modulators may be administered to subjects inconjunction with EPO. In another alternative method, the non-peptideEPO-R modulators may be administered to patients with diminished amountsof EPO.

[0102] Non-peptide EPO-R modulators may be administered alone ortogether with exogenous EPO, in which case the administration can besimultaneous or sequential, as will be appreciated by those in the art.EPO is usually administered by injection, whereas the non-peptide EPO-Rmodulators may be administered by a variety of routes as describedbelow.

[0103] The non-peptide EPO-R modulators may serve as EPO enhancers in amammal. In addition, because of the effect between non-peptide EPO-Rmodulators and EPO, the non-peptide EPO-R modulators may also be usedfor enhancing the physiological effect of EPO binding to EPO-R byadministration of non-peptide EPO-R modulators. Non-peptide EPO-Rmodulators may be used in methods for the diagnosis and therapy ofdiseases that involve inadequate or inappropriate EPO-R response.

[0104] Because the non-peptide EPO-R modulators may be used to replaceor enhance the effect of EPO, the non-peptide EPO-R modulators may beused to treat diseases caused by an insufficient amount of EPO or by alack of sufficient response to EPO, including hypoplastic anemia.

[0105] Anemia develops whenever there is a deficiency in erythrocytecount. It develops in response to various causes such as sicklingdisorders, homozygous beta thalassemia, hereditary spherocytosis, redcell enzymopathies, iron, vitamin B₁₂, folates deficiencies, aplasticanemia, Fanconi's anemia, Blackfan Diamond anemia, or leukemia. When apatient suffers from acute renal failure or when chronic renal failuredevelops, the inadequate renal production of erythropoietin results inhypoplastic anemia. Similarly, anemia in premature infants develops whena progressive fall in hemoglobin concentration, relatively low absolutereticulocyte counts, and bone marrow erythroid hypoplasia develops fromlow concentration of serum erythropoietin. Patients undergoing kidneydialysis frequently develop anemia.

[0106] The effect of reduced erythropoietin on development of anemiaduring chronic renal failure has been known and is further substantiatedby findings described in Lancet, 1175 (1986) and in N.Engl.J.Med, 316:731 (1987), which report that anemia in patients undergoing hemodialysisis completely reversed with recombinant human erythropoietin. Thesefindings suggest that the anemia is due at least in part toerythropoietin deficiency.

[0107] As used herein, “treatment” is an approach for obtainingbeneficial or desired results, including and preferably clinicalresults. Treatment can involve optionally either the amelioration ofsymptoms of a disease or condition, the prevention of symptoms of thedisease or condition, or the delaying of the progression of the diseaseor condition. Diseases and conditions that may be treated using thenon-peptide EPO-R modulators described in this application include butare not limited to the diseases and conditions described in thisapplication (e.g., includes any disease or condition ameliorated orpartially ameliorated by an increase in red blood cell level).

[0108] Because the subject compounds are not subject to rapiddegradation upon administering orally or parenterally, a wide variety offormulations may be employed. Pharmaceutical compositions comprising anon-peptide EPO-R modulator and a pharmaceutically acceptable carrier,diluent, or excipient are provided. It will be appreciated thepharmaceutical composition may optionally include one or more additionalcomponents. In this application, unless the context makes clearotherwise, we refer to carriers, diluents, and excipients collectivelyas “excipients.”

[0109] Generally, a pharmaceutical composition will include atherapeutically effective amount of a non-peptide EPO-R modulator and apharmaceutically acceptable excipient. The pharmaceutical compositioncomprising a non-peptide EPO-R modulator may be provided in a unitdosage form.

[0110] In this application, by “pharmaceutically acceptable” means thatthe carrier, diluent or excipient is compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Pharmaceutically acceptable excipients are well known in theart and include sterile water for pharmaceutical use, isotonic solutionssuch as saline and phosphate buffered saline, physiological saline, anddextrose solution. In addition, the pharmaceutical composition orformulation can include other carriers, adjuvants, or non-toxic,non-therapeutic, non-immunogenic stabilizers, excipients and the like.The compositions can also include additional substances to approximatephysiological conditions, such as pH adjusting and buffering agents,toxicity adjusting agents, wetting agents and detergents. Otherexcipients suitable for administration to a human patient are known inthe art. See, e.g., Remington: The Science and Practice of Pharmacy(20th edition, 2000, Gennavo, ed). The excipient is selected so as notto affect the biological activity of the agent or combination.

[0111] Depending upon the manner of administration of the non-peptideEPO-R modulators, the pharmaceutical compositions for delivery of thenon-peptide EPO-R modulators may be formulated in a variety of ways.

[0112] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

[0113] The components used to formulate the pharmaceutical compositionsare of high purity and are substantially free of potentially harmfulcontaminants (e.g., at least National Food (NF) grade, generally atleast analytical grade, and more typically at least pharmaceuticalgrade). For human consumption, the composition is manufactured orformulated under GMP standards. For example, suitable formulations maybe sterile and/or substantially isotonic and/or in full compliance withall Good Manufacturing Practice (GMP) regulations of the U.S. Food andDrug Administration.

[0114] In one version, the pharmaceutical composition comprises anon-peptide EPO-R modulator and a pharmaceutically acceptable carrier,where the non-peptide EPO-R modulator is selected from non-peptide EPO-Rmodulators described in this application. The subject compounds arenormally uniformly distributed in the pharmaceutical composition, are ina form that is physiologically available to the host, in beingtransported to the site(s) for treatment, and are at concentrations toprovide an effective dosage based on the manner of administration andthe frequency of application or ingestion.

[0115] For therapy, the non-peptide EPO-R modulators may be administeredby any means capable of delivering an effective amount of thenon-peptide EPO-R modulator. Non-limiting examples of administration ofthe non-peptide EPO-R modulators include oral, topical, or parenteraladministration, e.g. by injection at a particular site, for example,subcutaneously, intraperitoneally, intravascularly, intranasally,transdermally or the like. Formulations for injection will comprise aphysiologically acceptable medium, such as water, saline, PBS, aqueousethanol, aqueous ethylene glycols, or the like. Water solublepreservatives which may be employed include sodium bisulfite, sodiumthiosulfate, ascorbate, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric borate, parabens, benzyl alcohol andphenylethanol. Suitable water soluble buffering agents that may beemployed are alkali or alkaline earth carbonates, phosphates,bicarbonates, citrates, borates, acetates, succinates and the like, suchas sodium phosphate, citrate, borate, acetate, bicarbonate andcarbonate. Additives such as carboxymethylcellulose may be used as acarrier. The formulation will vary depending upon the purpose of theformulation, the particular mode employed for modulating the receptoractivity, the intended treatment, and the like. The formulation mayinvolve patches, capsules, liposomes, time delayed coatings, pills, ormay be formulated in pumps for continuous administration. The specificdosage can be determined empirically in accordance with known ways. See,for example Harrison's, Principles of Internal Medicine, 11th ed.Braunwald et al. ed, McGraw Hill Book Co., New York, 1987.

[0116] Generally, an “effective amount” or a “therapeutically effectiveamount” of a substance is that amount sufficient to effect a desiredbiological effect, such as beneficial results, including clinicalresults, and, as such, an “effective amount” depends upon the context inwhich it is being applied. In the context of administering a non-peptideEPO-R modulator to a vertebrate, human or other animal, atherapeutically effective amount may be an amount that can increasehematocrit levels in an animal (for example, an anemic human patient orother animal), e.g., an increase of at least about 10%, at least about20%, at least about 50%, and may be about 100%, or more. Alternatively,an effective amount may be an amount of non-peptide EPO-R modulatorsufficient to enhance the action of EPO in the human or non-humananimal. For example, for a patient in need of administration ofexogenous EPO (e.g., a patient with anemia) administration of aneffective amount of non-peptide EPO-R modulator will be able to reducethe amount of exogenous EPO required to achieve a given endpoint (e.g.,a designated level of erythrocytes in the animal, e.g., a normal orhealthy level). The reduction in the amount of exogenous EPO employed,as compared to the absence of a subject compound, may be at least about10%, often at least about 20%, sometimes at least about 50%, or more. Aneffective amount can be administered in one or more administrations inaccordance with a predetermined regimen.

[0117] The amount of non-peptide EPO-R modulators given to each subjectdepends on pharmacological properties such as bioavailability, clearancerate and route of administration. The dosage will also depend upon thebiological activity of the subject compound, the amount of EPO and/orother drugs being administered, the physiological condition of the host,and the like. Useful dosage ranges will be 0.01 to 200ug/kg, moreusually 0.05 to 100 ug/kg. Administration may be weekly or biweekly, oras often as daily, twice daily, more than twice daily, every two days,or less or more frequently, depending on the level of drug administered.

[0118] The host may be any mammal including domestic animals, pets,laboratory animals and primates, particularly humans. The amount willgenerally be adjusted depending upon the half life of the non-peptideEPO-R modulator, where dosages in the lower portion of the range may beemployed where the non-peptide EPO-R modulator has an enhanced half lifeor is provided as a depot, such as a slow release composition comprisingparticles, introduced in a matrix which maintains the non-peptide EPO-Rmodulator over an extended period of time, e.g., a collagen matrix, useof a pump which continuously infuses the peptide over an extended periodof time over a substantially continuous rate, or the like. Heller,Biodegradable Polymers in Controlled Drug Delivery, in: CRC CriticalReviews in Therapeutic Drua Carrier Systems, Vol. 1, CRC Press, BocaRaton, Fla., 1987, pp 39-90, describes encapsulation for controlled drugdelivery, and Di Colo (1992) Biomaterials 13:850-856 describescontrolled drug release from hydrophobic polymers.

[0119] The subject compounds are commercially available or may bereadily synthesized by common synthetic strategies.

[0120] The E5 and E5 analog molecules described above are available fromMaybridge, Plc (Cornwall, UK) and Key Organics Ltd (Cornwall, UK).

[0121] E5 may be synthesized as follows. There is literature precedentdescribed (J. Het. Chem., (1999), 36, 183-188), which is summarized inScheme 1, that will provide the core nucleus.

[0122] The following route (scheme 2) may then be used to synthesize theE5 target molecule.

[0123] The E4 molecule described above is available from one ofMaybridge, Plc (Cornwall, UK) and Key Organics Ltd (Cornwall, UK). Thecompound has been reported (London et al., J. Chem. Soc., (1939)902-906, and Miller et al., Synth. Comm., (1978) 371-376) and there isprecedent for the route of choice (Hamed et al., J. Chem. Soc., Perkin1, (1997) 2415-2422); the starting materials are commercial availableand the method of synthesis is shown in scheme 2 below.

[0124] The E6 molecule described above is available from one ofMaybridge, Plc (Cornwall, UK) or Key Organics Ltd (Cornwall, UK).

[0125] E6 may be synthesized as follows:

[0126] The precursor compound has been reported (Kaji et al., Chem.Pharm. Bull., (1970), 18, 147-156) and the synthesis uses commerciallyavailable starting material, as shown in scheme 3.

[0127] Ref. 5. Kaji, et al., 1970 Chem Pharm Bull18, 147-56

[0128] Ref. 6. Latti, et al., 1998 J Labelled Compounds Radiopharm 41,191-200

[0129] Generally, synthesis of analogs of E4, E5 and E6 may beaccomplished using techniques of synthetic organic chemistry known tothose in the art.

[0130] The compounds of the subject invention were shown to be active inbinding to EPO-R and in some instances activating EPO-R in culture andin vivo. The initial competitive ERP screening assay was designed as asolid plate-binding assay. A detailed protocol of the assay is describedbelow in the Protocolfor ERP Screen section.

[0131] Briefly, in the competitive ERP screening assay, theextracellular portion of EPO-R was purchased from R&D (Minneapolis,Minn.); biotinylated and regular peptide were synthesized by AmericanPeptide Company; receptor and biotinylated peptide (bio-ERP) wereincubated at room temperature; the solution was contacted withneutravidin (streptavidin) coated plate to bind any complex betweenreceptor and peptide; the complex that was bound to the plate isdetected by antibody specific for EPO-R and detection is performed withHRP-conjugated secondary antibody; the presence of the complex betweenreceptor and peptide is detected by light absorbance at 490 nm; and lackof or low signal indicates that the biotinylated peptide has beencompeted out, indicating an affinity of the test compound for themodulating domain of the EPO-R.

[0132] The library of compounds screened was created as a combination oftwo libraries: compounds were purchased from Maybridge Plc (Cornwall,UK) and Key Organics Ltd (Cornwall, UK). The libraries were received asmicrotiter plates. From the plates the compounds were diluted to 10_Mfinal concentration and used in a screen. Positive hits were identifiedas those compounds that were able to compete out the biotinylated-ERPpeptide in its binding to the target site on EPO-R. 80% of competitionwas use as a cut off criteria for positive binders. Hits were retestedin the binding assay to determine their dose-response curves.

[0133] Compounds identified in the initial screen were further testedfor their biological activity in two assays: a cell proliferation assayand a cell signaling assay in TF-1 and UT-7 cells. The hit rate in cellproliferation was 0.16%; hit rate in cell signaling was 0.25%.

[0134] At the end of the biological testing in cell lines TF-1 and UT-7,E4, E5, E5 analogs and E6 molecules were some of the compoundsidentified as showing the best activity. The activity was compared toEPO natural hormone and it was observed that E4, E5, E5 analogs, and E6molecules possess a maximum activity comparable to that of the maximumactivity of EPO in the presence of low levels of EPO.

[0135] In addition to the above assays, further in vivo studies wereundertaken (described in more detail below) including a Carboplatininduced anemia model, in which E4, E5, E5 analog, and E6 molecules werefound to restore hematocrit levels in animals treated with carboplatin;and erythroid colony formation (CFU-e) experiments with mouse and humanbone marrow, in which E4, E5, E5 analog, and E6 molecules were found tostimulate CFU-e formation to the same extent as EPO stimulates CFU-eformation.

[0136] The results of some of these assays are included in the figuresof this application.

[0137] The subject compounds can be provided as kits, particularly formarketing, comprising at least a subject compound and instructions forits use. The instructions may be written or electronic, using variouselectronic media, such as CD, floppies, tapes, etc. for theinstructions. The instructions may be labels provided with a therapeuticformulation, indicating dosages, side effects, acceptable andunacceptable combinations, and the like. For assays, the kits mayinclude at least the modulating domain of EPO-R, a labeled subjectcompound, cells comprising EPO-R or cells able to upregulate theexpression of EPO-R, and appropriate instructions as indicated above.

[0138] The following examples are intended to illustrate but not limitthe invention.

EXPERIMENTAL

[0139] The initial competitive ERP screening assay was designed as asolid plate binding assay. A detailed protocol of the assay is describedbelow in the Protocolfor ERP Screen section.

[0140] Briefly, in the competitive ERP screening assay, theextracellular portion of EPO-R was purchased from R&D (Minneapolis,Minn.); biotinylated and regular peptide were synthesized by AmericanPeptide Company; receptor and biotinylated peptide (bio-ERP) wereincubated at room temperature; the solution was contacated withneutravidin (streptavidin) coated plate to bind any complex betweenreceptor and peptide; the complex that was bound to the plate isdetected by antibody specific for EPO-R and detection is performed withHRP-conjugated secondary antibody; the presence of the complex betweenreceptor and peptide is detected by an light absorbance at 490 nm; andlack of or low signal indicates that the biotinylated peptide has beencompeted out, indicating an affinity of the test compound for themodulating domain of the EPO-R.

[0141] The library of compounds screened was created as a combination oftwo libraries: compounds were purchased from Maybridge Plc (Cornwall,UK) and Key Organics Ltd (Cornwall, UK). The libraries were received asmicrotiter plates. From the plates the compounds were diluted to 10 uMfinal concentration and used in a screen. Positive hits were identifiedas those compounds that were able to compete out the biotinylated-ERPpeptide in its binding to the target site on EPO-R. 80% of competitionwas use as a cut off criteria for positive binders. Hits were retestedin the binding assay to determine their dose-response curves.

[0142] Compounds identified in the initial screen were further testedfor their biological activity in two assays: a cell proliferation assayand a cell signaling assay in TF-1 cells. The hit rate in cellproliferation was 0.16%; hit rate in cell signaling was 0.25%.

[0143] At the end of the biological testing in cell line, E4, E5, E5analog, and E6 molecules were some of the compounds identified asshowing the best activity. The activity was compared to EPO naturalhormone and it was observed that E4, E5, E5 analog, and E6 moleculespossess a maximum activity comparable to that of the maximum activity ofEPO.

[0144] In addition to the above assays, further in vivo studies wereundertaken (described in more detail below) including a Carboplatininduced anemia model, in which E4, E5, E5 analog, and E6 molecules werefound to restore hematocrit levels in animals treated with carboplatin;and erythroid colony formation (CFU-e) experiments with mouse and humanbone marrow, in which E4, E5, E5 analog, and E6 molecules were found tostimulate CFU-e formation to the same extent as EPO stimulates CFU-eformation.

[0145] The results of some of these assays are included in the figuresof this application.

[0146] Protocol for ERP Screen

[0147] Candidate molecules were screened for EPO-R binding. Compoundswere diluted in minisorb plates 1:20. 10 ul of the compound solution wasadded with matrix impact electronic multi pipettor into each well of theplate. 10 ul of cold peptide (with 5% DMSO) was then added (or HBS incl.5% DMSO to control wells). ERP was diluted 1:4(25 μl 1 mM+75 uL HBS, 250uM) and put on ice. 26 μl of a 2 uM solution of EPO receptor was addedto a receptor tube containing 2054 ul of HBS and vortexed a little. 20ul of this solution was added to each well with repeat pipettor per well(10 nM in competition); 8.8 μl of a 250 uM solution of ERP-Biotin wasadded per tube with 2.2 ml HBS, and vortexed a little. 20 ul of thissolution was dispensed with repeat pipettor into wells (400 nM incompetition). The plates were then incubated for 90 min. at roomtemperature (RT) on a horizontal shaker. 150 ul of HBS with 0.2% BSA and2% Tween-20 was added to the wells. The plates were then incubated for60 minutes at RT. 50 ul/well were transferred to a Neutravidin plate andincubated for 30 min at RT on a shaker. The wells were washed with 3×HBS0.05% Tween-20 and hit on towels after last wash. Anti-EPO-R ab (m.c.R&D) was added diluted 1:400 in HBS +0.3% Dry Milk +0.05% Tween-20repeat pipettor plus. The wells were incubated for 1 hour at RT in ahorizontal shaker. The wells were washed 3 times with HBS 0.05% Tween-20and then hit on towels after last wash. Anti-mouse Ab-HRP (Santa Cruz)was diluted 1:1000, in HBS +0.3 % Dry Milk +0.05% Tween 20 and added tothe wells. Following a 30 min RT incubation, the wells were washed 3times with HBS 0.05% Tween-20. 100 ul TMB (N,N,N′,N′-tetramethylbenzidine) was added and after 15 minutes of RT incubation, 50 ul 2Msulfuric acid was added. The absorbency of the wells was read at 450 nm.

[0148] Stock solution aliquots: EPO-R at 2 μM (50ug/ml PBS) 300ul/siliconized tube, 60 tubes; ERP-Biotin 50 μl at 1 mM 60 tubes; ERP120 ul aliquots 60 tubes 1 mM water; make 10×HBS.

[0149] Solutions needed for each day: HBS with 2% Tween 20 and 0.2% BSA(500 ml): 10 g Tween-20; 1 g BSA (SIGMA); fill up to 500 ml with HBS;Wash buffer (HBS with 0.05% Tween 20) 1.21 10×HBS; plus 10.81 water;plus 6 g Tween-20 (put Tween first in little beaker dissolve in somewater, integrate into volume of water added to the 10×HBS.

[0150] Assay buffer: 500 ml Wash buffer plus 0.3% (1.5g) Safeway DryMilk;

[0151] Cell proliferation:

[0152] TF-1 cells were stimulated for 96 hours with differentnon-peptide EPO-R modulators, and it was observed that the non-peptideEPO binding molecules stimulated cellular proliferation indose-responsive manner and to the extent of the natural hormone. Resultsdemonstrated that non-peptide EPO-R modulators have anti-apoptoticeffect on cells by acting through EPO-R. (See FIG. 3)

[0153] Growth and Starvation of UT-7 Cells

[0154] UT-7 cells were grown in Minimal Essential Medium Alpha (MEM-α)containing 20% FBS, 1.5 g/L NaHCO₃, 5 ng/ml GM-CSF, and 100UPenicillin/100 ug/ml Streptomycin Sulfate (“P/S”), in T150 flasks (60ml/flask) at 37° C., 5% CO₂ to a density of no more than 5×10⁵ cells/ml.The cells were starved overnight in MEM-α containing 10% FBS, 1.5 g/LNaHCO₃ and P/S in T150 flasks (60ml/flask) at a density of 5×10⁵cells/ml, in 5% CO₂, 37° C. The cells were then centrifuged in aswinging-bucket rotor at 200×g for 5 minutes at room temperature. Thesupernatant was removed, and the cells pooled into one 50 ml conicaltube. The cells were washed twice with serum-free medium (MEM-alphacontaining 1.5 g/L NaHCO₃ and P/S). After the last wash, the cells wereresuspended to a density of 1×10⁷ cells/ml in serum-free medium. Thecells were then placed in 15 ml conical tubes in 1 ml aliquots andincubated with the caps loosened for 1 hour at 37° C., 5% CO₂. Every 10minutes, the lower portion of each tube was tapped (flicked) severaltimes to prevent the cells from settling (please note, this step is veryimportant!)

[0155] In vitro Signaling:

[0156] TF-1 or UT-7 cells were stimulated for up to 30 minutes andactivation of EPO-Receptor down stream signaling pathway was evaluated,i.e. phosphorylation of JAK2, STAT1, STAT3 and STAT5. Resultsdemonstrated that non-peptide EPO-R modulators activated EPO-R signaltransduction. It was also observed that EPO-R was phosphorylated whenstimulated with non-peptide EPO-R modulators. (See FIG. 4)

[0157] Signaling in TF-1 Cells in Response to EPO and Compounds

[0158] Cell Growth and Starvation: TF-1 cells were grown in RPMI 1640medium containing 10% FBS, 2 ng/ml human GM-CSF, and 100 UPenicillin/100 ug/ml Streptomycin Sulfate (“P/S”), in T150 flasks (60ml/flask) at 37° C., 5% CO₂. The cells were grown to a density of nomore than 8×10⁵ cells/ml. The cells were starved overnight in RPMI 1640medium containing 3.5% FBS, and P/S in T150 flasks (60 ml/flask) at adensity of 8×10⁵ cells/ml, in 5% CO₂, at 37° C. On the day of the assay,before processing the cells, the non-peptide EPO-R modulator to betested was diluted as described below. The diluted compounds sit at roomtemperature for 90 min before being added to the cells. The cells werecentrifuged in a swinging-bucket rotor at 200×g for 5 min at roomtemperature. The supernatant was removed and the cells were pooled intoone 50 ml conical tube. The cells were washed twice with serum-freemedium (RPMI 1640 containing P/S). After the last wash, the cells wereresuspended to a density of 1.5×10⁷ cells/ml in serum-free medium. Thecells were placed in 15 ml conical tubes in 1 ml aliquots, and incubatedwith the caps loosened for 1 hour at 37° C., 5% CO₂. Every 10 minutes,the lower portion of each tube was tapped several times to prevent thecells from settling.

[0159] Cell Activation and Immunoprecipitation

[0160] While the cells were starving, GammaBind G Sepharose beads werecoated with antibody. To do this, the GammaBind G beads were washed 3times with PBS (the beads come in a slurry containing 50% beads; 30 ulof this slurry was used per sample). First, the slurry was spun for 15to 20 seconds, and the supernatant discarded. For each wash, 1 ml icecold PBS was added to the beads, and the tube inverted several times.The tube was spun for 15 to 20 seconds, and the supernatant discarded.After the last wash, the supernatant was discarded and 2 ug (10 ul)anti-phosphotyrosine antibody (PY99) per sample was added to the beads.Next, enough PBS was added so that the tube contains 50% beads and 50%liquid (for example, for 10 samples, 300 ul slurry was used; this givesapproximately 150 ul beads). The beads were washed and 100 ul antibodyplus 50 ul PBS were added. The beads were incubated for 2 hours at roomtemp with end-over-end rotation.

[0161] When the cells finished their 1 hour incubation, they werestimulated as follows: The cells receiving the non-peptide EPO-Rmodulator have a final DMSO concentration of 0.3%. To maintainequivalent conditions for all cells, DMSO was added to all of the cellsthat do not receive compounds. To obtain a final concentration of 0.3%DMSO, 10 ul 30% DMSO was added to 1 ml cells and mixed well. 1 ml ofcells containing 0.3% DMSO was used as a negative control, and 1 ml ofcells containing 300 U/ml EPO and 0.3% DMSO was used as a positivecontrol.

[0162] Dilutions: The samples were diluted to 100× their finalconcentration and 10 ul of each dilution was added to 1 ml of cells toobtain the final concentration. To obtain a final concentration of 30 uMERP, 30 ul of the 1 mM stock solution was added directly to 1 ml ofcells. For the dilutions, see the table below: Final Conc. 100xDilutions 300 mU/ml EPO   30 U/ml 6 ul 500 U/ml EPO stock + 94 ul icecold PBS  30 uM Compound   3 mM 9 ul 10 mM Compound stock + 21 ul H₂O  3uM Compound  0.3 mM 3 ul 3 mM Compound + 27 ul 27% DMSO  0.3 uM Compound0.03 mM 4 ul 0.3 mM Compound + 36 ul 27% DMSO  30 uM EPO-R — Add 30 μl 1mM EPO-R stock directly to 1 ml cells  3 uM EPO-R  0.3 mM 6 ul 1 mMEPO-R stock + 14 ul ice cold H₂O 3 ul 0.3 mM EPO-R (above) + 27ul icecold  0.3 uM EPO-R 0.03 mM H₂O

[0163] The samples were incubated for 30 minutes at 37° C., 5% CO₂, andevery 10 minutes, the samples were mixed by tapping (flicking) the lowerportion of each tube several times. The assays were stopped by adding 14ml of ice cold PBS to each sample. The samples were centrifuged at400×g, 4° C. for 5 minutes in a swinging bucket rotor. The samples wereplaced on ice, and the supernatant was aspirated. To lyse the cells, 800ul 2×lysis buffer (containing 2×protease inhibitors, added just beforeuse) was added to each cell pellet and pipeted up and down severaltimes. The cells were then incubated on ice for 30 minutes. The sampleswere transferred into 1.5 ml microcentrifuge tubes, and spun for 10minutes at 10,000×g, 4° C. While the samples were spinning, theantibody-coated GammaBind G beads were separated into equivalentaliquots in an appropriate number of 1.5 ml microcentrifuge tubes. Thesupernatant was taken from each sample, and added to theanti-phosphotyrosine-coated GammaBind G beads, and the beads andsupernatant mixture were incubated with end-over-end rotation over nightat 4° C. The samples were spun for 15 to 20 seconds, and the supernatantwas discarded. The beads were washed twice with 800 ul 1×lysis buffer(containing 1×protease inhibitors, added just before use) and once with800 ul of a 1:1 mix of lysis buffer: 125 mM Tris pH 6.8 (for each wash,add buffer, invert the tube several times, spin for 15 to 20 seconds,and discard the supernatant). 45 ul 1×sample buffer was added to eachsample. The samples were heated for 5 minutes at 95° C., centrifugedbriefly (approx. 20 seconds) and examined by SDS-PAGE.

[0164] Western Analysis

[0165] The samples were run on two 8% SDS-Polyacrylamide gels underreducing conditions. The procedure was to transfer the proteins toImmobilon-P membranes (transfer for 60 min., at 300 mA, in 10 mM CAPStransfer buffer, pH 11 containing 10% MeOH); block the membranes inblotto for 1 hr at room temp; incubate one membrane with alpha-STAT 5bantibody, and the other with alpha-EPO-R antibody (dilute bothantibodies 1:1,000 in blotto) at 4° C. overnight; wash the membranes 3times in blotto at room temp., 4 minutes per wash; add alpha-Rabbit-APsecondary antibody diluted 1:2,000 in blotto to both membranes; incubatefor 2 hours at room temp; wash the membranes 3 times with blotto, 4minutes per wash; wash the membranes twice with 1×TST, 4 minutes perwash; wash the membranes once with 1×TSM, 4 minutes per wash; add 10 mlBCIP/NBT substrate to each membrane for detection.

[0166] Solutions

[0167] 2×Lysis Buffer: 100 mM HEPES, pH 7.6  50 ml 1 M HEPES, pH 7.6 300mM NaCl 8.8 g NaCl 2% Triton X-100  10 ml Triton X-100  10 mM EDTA  10ml 0.5 M EDTA, pH 8

[0168] 100×Protease Inhibitors:   1 mg/ml Aprotinin  20 mg Aprotinin 100 ug/ml Pepstatin A*  2 mg Pepstatin A*  100 ug/ml Leupeptin  2 mgLeupeptin  100 ug/ml Chymostatin*  2 mg Chymostatin* 23.8 mg/ml AEBSF476 mg AEBSF

[0169] Sample Buffer:

[0170] 100 ul 50% Glycerol,

[0171] 0.05% Bromphenol Blue

[0172] 20 ul u-Mercaptoethanol

[0173] 40 ul 10% SDS

[0174] 160 ul 1×gel running buffer (electrophoresis buffer)

[0175] 10×TST: 0.1M Tris pH 7.4 12.1 g Tris base 1.5M NaCl 87.7 g NaCl0.75% Tween-20  7.5 ml Tween-20  0.2% NaN₃   3 g NaN₃

[0176] Blotto: 1x TST 100 ml 10x TST 5 g Dry Milk 0.5% Dry Milk

[0177] 10×TSM: 1M Tris pH 9.0 121.1 g Tris base 1M NaCl  58.4 g NaCl 50mM MgCl₂  10.2 g MgCl₂ × 6H₂O

[0178] When the subject compounds were tested for anti-apoptotic effectwas evaluated, these antibodies were used: Anti-Bcl-XL/s antibody (SantaCruz Biotechnology, SC-1041), Anti-Bcl-X_(L) antibody (Santa CruzBiotechnology, SC8392)

[0179] BCIP/NBT Substrate:

[0180] NBT stock: 50 mg/ml in 70% Dimethyl Formamide. Store at −20° C.;BCIP stock: 50 mg/ml in 100% Dimethyl Formamide. Store at −20° C.; Justbefore using, combine 10 ml 1×TSM with 66 ul 50 mg/ml NBT, and 33 ul 50mg/ml BCIP; Mix well, and apply to filter.

[0181] Materials:

[0182] Anti-EPO-R Antibody (c-20): Santa Cruz Biotech, cat. #sc-695;Anti-STAT 5b Antibody (c-17): Santa Cruz Biotech, cat. #sc-835;Anti-Phosphotyrosine Antibody (PY99): Santa Cruz Biotech, cat. #sc-7020;Anti-Rabbit IgG-AP: Santa Cruz Biotech, cat. #sc-2007; BCIP(5-Bromo-4-Chloro-3-Inoloyl Phosphate): Sigma, cat. #B-8503; CAPS Buffer(3-[Cyclohexylamino]-1-propanesulfonic acid): Sigma, cat. #C-2632; FetalBovine Serum: American Type Culture Collection (ATCC), cat. #30-2020;GammaBind® G Sepharose: Pharmacia Biotech, cat. #17-0885-02; HumanGM-CSF: PeproTech, cat. #300-03; Immobilon P: Millipore, cat.#IPVH00010; NBT (Nitro Blue Tetrazolium): Sigma, cat. #N-6876; PBS(Phosphate Buffered Saline): Sigma, cat. #P-3813;Penicillin/Streptomycin Sulfate: Applied Scientific, cat. #9366; RPMI1640: American Type Culture Collection (ATCC), cat. #30-2001;

[0183] Formation of Erythroid Colonies (CFU-e, BFU-e) from Bone Narrow:

[0184] Mouse or human bone marrow cells were stimulated inmethylcellulose for 7 days (mouse) or 14 days (human) with non-peptideEPO-R modulators. Formation of erythroid colonies was observed. (SeeFIGS. 6 to 8)

[0185] Protocol for Human Bone Marrow Isolation

[0186] (1) Dilute human bone marrow 1:1 with sterile PBS. (2) Slowlylayer bone marrow into 50 ml tubes containing 20 ml of LSM Lymphocyteseparation medium (ICN/Cappel—cat #50494). 50 ml bone marrow will yield2 tubes. (3) Spin tubes at 2000 rpm for 30 minutes with brake off. (4)Slowly take out the middle layer using a 10 ml pipette and transfer intoa new 50 ml tube. (5) Add PBS +0.5% BSA +5 mM EDTA to tube for washing.(6) Spin tube for 5 minutes at speed 4 with brake on. (7) Repeat washstep 2 more times. (8) After 3^(rd) wash, resuspend pellet in SFIscoves' MDM medium. (9) Count by diluting cells in 3% acetic acid. (10)Dilute cells to desired concentration (5×10⁵ cells/ml) and seed ontomethylcellulose.

[0187] Protocol for Human Bone Marrow Seeding in SemisolidMethylcellulose Medium

[0188] (1) Tubes containing 3.0 ml of methylcellulose medium (StemcellTechnology, cat #H4230) supplemented with 50 ng/ml hSCF are prepared.(Alternatively the methylcellulose could be supplemented with 50 ng/mlhSCF, 20 ng/ml of hIL-3 and hIL-6). (2) Compounds or EPO of desiredconcentrations are added to the tubes. “Control” tubes or tubes thatwill receive EPO are supplemented with DMSO to a final concentration of0.16%. The same DMSO concentration is kept constant in all otherexperimental conditions. (3) 300 ul of human bone marrow cells at 5×10⁵cells/ml is added to each tube, vortexed briefly, and the tubes allowedto sit in a test tube rack for 5 minutes. 1.1 ml of the cell mixture isaliquoted into two 35 mm dishes using a 16 g blunt end needle (StemcellTechnology). A third 35 mm dish containing 3 ml of sterile water isplaced together with the two dishes into a 125 mm tissue culture dish.(4) The dishes are incubated at 37° C., 5% CO₂ and observed periodicallyfor colony formation. Colonies are scored on day twelve.

[0189] Colonyformation Assay

[0190] Bone marrow cells were isolated from mouse femurs. Cells werewashed twice with PBS and resuspended to a density of 6.5×10⁶ cells/mlin media. 300 ul (6.5×10⁵/ml) of cells were mixed with 1.5 ml ofmethylcellulose media (Stemcell Technology) to which the desiredconcentration of compound or the EPO was added. The mixture was pouredonto 35 mm dishes; all the conditions are performed in duplicates. Theincubation was for eight days at 37° C., 5% CO₂. The colonies werescored on day seven.

[0191] Materials:

[0192] Stemcell Technology: 1. Methycellulose base medium for murinecells cat #M3234 80 ml/bottle; 2. 35 mm dish cat #27150 500/pack cat#27100 10/pack; 3. Iscove's Modified Dulbecco's Medium (MDM) with 2% FBScat #07700 100 ml/bottle; 4. 16 gauge blunt-end needle cat #28110 packof 100; 5. 3 cc syringes cat #28240 pack of 1000; 6. Gridded scoringdishes cat #27500 5/pack;

[0193] Applied Scientific: 1. minisorp tube 100×15 cat #468608; 2.minisorp tube 100×5 stopper cat #343036; 3. 10 cc syringe cat #9604;

[0194] Corning: 1. sterile 100 mm tissue culture dish cat #25020;

[0195] Additional materials: 1. sterile water; 2. counter.

[0196] After thawing methylcellulose overnight, the next morning, 20 mlof Iscove's MDM+2% FBS is added to the bottle to achieve 100 ml total.The bottle is mixed for at least 30 seconds and settled for about 10minutes. 10 cc syringes are used to aliquot 3 ml per minisorp tube.

[0197] CFU-e A Formation From Fetal Liver Cells

[0198] Mouse fetal liver cells were isolated and stimulated inmethylcellulose with non-peptide EPO-R modulators for 3 days. Formationof erythroid colonies was observed. (See FIG. 5)

[0199] Mouse Fetal Liver CFU-e Assays

[0200] The procedure was as follows. 1. Remove fetuses from 1 to 2 E12Timed pregnant CD-1 mice (10-12 fetuses; Charles River) into ice coldIMDM (StemCell Tech) in a 10-cm dish; 2. Dissect out fetal liver (FL)from fetuses in IMDM under a dissection scope; 3. Wash FL 2× with IMDM;4. Add 1-2 mL IMDM; disaggregate the FL into cell suspension by pipetingup/down with a P1000/blue tip 10-15 times; 5. Pass the cell suspensionthrough a Cell Strainer (70 u, Falcon 352350); 6. Wash cells 3× in IMDM;Resuspend cells in 1 mL IMDM/2% FBS (StemCell) and count nucleated cells1/20 in 3% acetic acid; 12 FL should yield around 4×10⁷ nucleated cells;7. Dilute FL cells in IMDM/2% FBS to 0.5-1×10⁶/mL; 8. Add 0.3 ml cellsinto 3 mL 1×M3231 (StemCell Technologies) containing recombinant humanerythropoietin alpha and/or other cytokines/compounds; 9. Vortexvigorously and let stand for 5 minutes; 10. Dispense 2×1.1 mL into 35-mmdishes (StemCell #27100) using a 16-gauge blunt-end needle/3 mL syringe;11. Place the 2 35-mm dishes into a 10-cm culture dish along with athird uncovered 35-mm dish containing 3 mL sterile water; 12. Incubate2-3 days at 37C./5% CO₂ and score for CFU-e.

[0201] Optional: Benzidine Staining of CFU-e/BFU-e

[0202] 1. Make benzidine staining soln (make up fresh)--0.2% BenzidineHCl (Sigma B-3383), 0.5% glacial acetic acid, 0.12% H₂O₂ (0.2 mL/50 mL30% H₂O₂, Sigma H-1009); 2. Gently add sufficient amount (0.75 mL/35 mmdish) of benzidine staining solution into Methocult dishes to cover thecells; Wait a few minutes; cells expressing hemoglobin will turn blue.

[0203] In vivo Experiments with Anemic Animals

[0204] Mice were treated with Carboplatin agent (available from Sigma,used regularly in chemotherapy) and a drop in hematocrit was induced.Intraperitoneal (ip) or oral administration of non-peptide EPO-Rmodulators protected from a drop in hematocrit levels of Carboplatintreated animals; i.e. at least partially prevented development ofanemia. (FIGS. 9-11)

[0205] Determination of Effect of Non-peptide EPO-R Binding SmallMolecule on Hematocrit in Carboplatin-Induced Anemia in 8 Week C57BIMale Mice

[0206] Injection Protocol

[0207] Preparation of injection solutions: Vehicle—Saline with 0.1 mg/mLBSA; Add 30 mg of BSA to 300 mL Saline; Vacuum filter under sterileconditions in laminar flow hood; Aliquot the saline (0.1 mg/mL BSA)solution into mini-sorp tubes. At least 41×7 mL aliquots should beprepared. Place caps on the tubes and store refrigerated at 4° C.;

[0208] Carboplatin—Dose of 120 mg/kg: Calculations for single injectiongiven to 36 male mice (8 wks old˜25 g): 120 mg/mouse×0.025 kg×36mice=108 mg; Injections are delivered i.p. in a volume of 0.5cc/mouse=0.5 cc/mouse×36 mice=18 cc; Carboplatin at 6 mg/mL for 36 mice:108 mg/18 cc.; 120 mg/20 cc; On day of injection, add 120 mg carboplatin(Sigma #C2538) to 20 cc saline in a 100 cc beaker containing a stirringbar. Cover beaker with parafilm. Mix until the carboplatin hascompletely dissolved.

[0209] Non-peptide EPO-R Binding Small Molecule—Dose at 11 nmole/kg/Day

[0210] Calculations for daily injections given to 12 male mice (8 wksold˜25 g) over a period of 10 days:

[0211] 11 nmole/kg/mouse/day×0.025 kg×12 mice=3.3 nmoles/day; Injectionsare delivered i.p. in a volume of 0.5 cc/mouse/day=0.5 cc/mouse/day×12mice=6 cc/day; non-peptide EPO-R binding small molecule for 20 mice: 3.3nmoles/6 cc each day: 3.85 nmole/7 cc each day; non-peptide EPO-Rbinding small molecule aliquot at 2 mM=2 nmole/uL; 3.85 nmole per day in7 cc (0.55 nmole/mL); Therefore: 3.85 nmole divided by 2 nmole/uLstock=1.925 uL at 2 mM; Prepare in advance at least 10 aliquots ofnon-peptide EPO-R binding small molecule at 2 mM in small microfugetubes, each with a volume of 2 uL. Freeze these aliquots; On each day ofinjections, add 1 thawed aliquot of the non-peptide EPO-R binding smallmolecule at 2 mM containing a 2 uL volume to a tube containing 7 cc ofsterile saline (0.1 mg/mL BSA). This results in an non-peptide EPO-Rbinding small molecule injection solution with 0.55 nmole/mLconcentration.

[0212] Erythropoietin Dose at 1.5 ug/kg/day: Calculations for dailyinjections given to 12 male mice (8 wks old˜25 g) over a period of 10days: 1.5 ug/kg/mouse/day×0.025 kg×12 mice=0.45 ug/day; Injections aredelivered i.p. in a volume of 0.5 cc/mouse/day=0.5 cc/mouse/day×12mice=6 cc/day; EPO for 12 mice: 0.45 ug/ 6 cc each day: 0.525 ug/7 cceach day; Reconstitute 500 units (5 ug) of EPO (#287-TC-500, R&DSystems) with 1 mL PBS to provide a stock concentration at 5 ug/mL; Use0.525 ug per day in 7 cc (75 ng/mL); Therefore: 0.525 ug divided by 5ug/mL stock=0.105 mL at 5 ug/mL˜100 uL at 5 ug/mL; Prepare in advance atleast 10 aliquots of EPO at 5 ug/mL in small microfuge tubes, each witha volume of 100 uL. Freeze these aliquots; On each day of injections,add 1 thawed aliquot of the EPO at 5 ug/mL with a volume of 100 uL to atube containing 7 cc of sterile saline (0.1 mg/mL BSA). This results inan erythropoietin injection solution with a concentration of 75 ng/mL.

[0213] Injection Regimen

[0214] Carboplatin Only Negative Control Group: Each animal is given a0.5 cc i.p. injection of carboplatin at 6 mg/mL on day 0. Each animal isgiven a 0.5 cc i.p. injection of saline (0.1 mg/mL BSA) on days 1through 10.

[0215] Injection Regime

[0216] Carboplatin+non-peptide EPO-R binding small molecule (11nmole/kg/day) Test Group: Each animal is given a 0.5 cc i.p. injectionof carboplatin at 6 mg/mL on day 0. Each animal is given a 0.5 cc i.p.injection of non-peptide EPO-R binding small molecule (0.55 nmole/mL) ondays 1 through 10.

[0217] Carboplatin+Erythropoietin (1.5 ug/kg/day) Positive ControlGroup: Each animal is given a 0.5 cc i.p. injection of carboplatin at 6mg/mL on day 0. Each animal is given a 0.5 cc i.p. injection ofErythorpoietin at 75 ng/mL on days 1 through 10.

[0218] Saline Control Group: Each animal is given a 0.5 cc i.p.injection of saline (0.1 mg/mL BSA) on day 0. Each animal is given a 0.5cc i.p. injection of saline (0.1 mg/mL BSA) on days 1 through 10.

[0219] Reticulocyte Measurement in Normal Mice

[0220] Normal mice were injected ip with non-peptide EPO-R modulators onday 0. Reticulocyte levels were measured on days 5, 8, and 11 afterinjections. Non-peptide EPO-R modulators increased reticulocyte levelsto the same extent as the natural hormone.(See FIG. 12)

[0221] Small molecules that act as a replacement and in cooperation withEPO were evaluated for their potential application in CNS. The resultsobtained indicated that indeed, the subject small molecules have aprotective role in neuronal apoptosis. The subject small moleculestested protected the cultured neurons from glutamate neurotoxicity byincreasing the cell survival by 60%. Thus, the subject small moleculescan find use in prevention of cerebral ischemia, spinal cord injury ormetabolic stress.

[0222] A number of molecules of the subject invention were evaluated inin vitro systems that reflect the condition of cerebral ischemia andmetabolic stress. Firstly, it is known that one of the mechanisms bywhich EPO prevents neuronal apoptosis is by up-regulating the expressionlevels of Bcl-x₁ protein, which is a well-described anti-apoptoticprotein. Thus, cells-lines that are known to be EPO responsive (UT-7 andTF-1, used in previous studies) were used as a cellular system toexamine the small molecules' anti-apoptotic activity through EPO-R.

[0223] Application of Subject Small Molecules in CNS System

[0224] As presented in FIGS. 13-14, in both cell lines, small molecules(E5A24 and EM5) induced up-regulation of Bcl-x_(L) protein in adose-responsive manner. Achieved up-regulation of Bcl-x_(L) protein wasto the levels achieved with EPO. The evaluation was performed bystarving the cells, stimulating the cells with compounds at appropriateconcentrations (or no stimulation for control samples) andimmunoprecipitating the cell lysates with antibody specific to bothforms (short and long) of Bcl-x_(L) protein. The amount of Bcl-x_(L)(long protein form, known to have anti-apoptotic activity) protein wasevaluated by western blot analysis.

[0225] Experiments were performed according to the described protocol.The anti-apoptotic effect of the subject small molecules was evaluatedin P-19 cells (a neural-like embryonal carcinoma cell line) that undergoapoptosis upon withdrawal of serum. Briefly, cells were treated withsmall molecules for 24 hours prior to serum withdrawal. Serum waswithdrawn for 48 hours in the presence or absence of a subject smallmolecule (EM5 and E5A24). At the end of that period, cell survival wasevaluated. As presented on FIG. 15, small molecules, in adose-responsive manner, protect cells from apoptosis. This Figure isrepresentative of the experiment and of the subject small molecules. Theexperiments were performed according to literature procedures (Siren et.al., Proc. Nat. Acad. Sci. 98, 4044, 2001). Cell survival was determinedaccording to the protocol described for cellular proliferation.

[0226] Primary cultured cells were used as a model to evaluate thesubject small molecule effect on CNS. Glutamate neurotoxicity can bemost directly studied using neurons prepared from the brain at the lateembryonic stage and subsequently cultured for maturation. To examine theeffect of the subject small molecules (E5A24 and E5A29) onglutamate-induced neuronal death, cortical neurons isolated fromembryonic day 18 rats, were cultured for 10 days. Subsequently, neuronswere treated for 24 hours with or without the subject small molecules,and further exposed to 300 uM glutamate for 24 hours. After glutamatewas washed out, the cells were cultured for another 24 hours. During theglutamate challenge and subsequent culture, the subject small moleculeswere absent. Finally, the cell viability was examined. FIG. 16 showsthat subject small molecules will increase cell survival by 60%. Thus,small molecules prevented glutamate induced neuronal death in adose-responsive manner. Experiments were performed according to theliterature (Morishita et. al., Neuroscience 76, 105, 1997).

[0227] The above results demonstrate the extraordinary capability of thesubject family of compounds to bind specifically to the modulationdomain of EPO-R with high affinity. By using non-peptide compounds thathave significant stability under a variety of conditions, includingphysiological and cellular conditions, one can purify EPO-R, assay forchanges in the amount of EPO-R present, particularly at a cell surface,in response to changes in environment, e.g. test compounds, withoutinterfering with binding of a ligand. One can also determine whether atest ligand and a subject compound act together to enhance the activityof EPO-R signal transduction, with concentrations of the EPO-R ligandbelow saturation.

[0228] In addition, subject compounds have been found to be effectivewith a variety of cells that express EPO-R inducing a signal into thecell upon binding to the modulating domain of the EPO-R. The subjectcompounds may be used individually or in combination with each other orEPO in inducing a signal.

[0229] The subject compounds, individually or in combination with EPOcan be used for enhancing hematocrit, allowing for reduced levels of EPOto achieve the same enhancement of hematocrit. The compounds were foundsafe with an exemplary laboratory animal, there being no observed toxicevents, while at the same time being able to correct induced anemia.

[0230] The subject compounds can be used with neuronal cells to modulateapoptosis. Their small size and lipophilicity enhance their ability tocross the blood-brain barrier. When endogenous EPO is deficient, thesubject compounds will serve to protect the neuronal cells. In arecognized test, the subject compounds were found to enhance survival ofcells in a neurotoxic environment. As EPO-R is found on other cells indifferent contexts, the subject compounds can find use in modulating theresponse of the cells in relation to a context that is affected by thepresence or absence of EPO.

[0231] Because the subject compounds are not readily degraded in theintestinal tract or in the blood, the subject compounds can beadministered orally, as well as parenterally. In addition, the subjectcompounds can be used for investigating other compounds, such asbiosteres, that have analogous effects with EPO-R, but may enjoyimprovements in bioavailability, potential side effects, idiosyncraticresponses and higher affinity, resulting in lower dosages.

[0232] The subject compounds can be readily prepared in high yieldfollowing known synthetic procedures. They share similarities with knowndrugs having physiological activity, so they can be readily formulatedin accordance with known methodologies. They find application wheneverand wherever one wishes to modulate EPO-R activity.

[0233] All references referred to in the text are incorporated herein byreference as if fully set forth herein. The relevant portions associatedwith this document will be evident to those of skill in the art. Anydiscrepancies between this application and such reference will beresolved in favor of the view set forth in this application.

[0234] Although the invention has been described with reference to theabove examples, it will be understood that modifications and variationsare encompassed within the spirit and scope of the invention.Accordingly, the invention is limited only by the following claims.

1 2 1 23 PRT Homo sapiens 1 Gln Arg Val Glu Ile Leu Glu Gly Arg Thr GluCys Val Leu Ser Asn 1 5 10 15 Leu Arg Gly Arg Thr Arg Tyr 20 2 23 PRTHomo sapiens 2 Gln Arg Val Glu Val Leu Glu Gly Arg Thr Glu Cys Val LeuSer Asn 1 5 10 15 Leu Arg Gly Gly Thr Arg Tyr 20

What is claimed is:
 1. A combination comprising a polypeptide comprisingthe modulating sequence of the erythropoietin receptor and a non-peptideorganic molecule of from 12 to 36 atoms other than hydrogen, from 9 to20 carbon atoms, and from 4 to 12 of the heteroatoms chalcogen,nitrogen, halogen, and metal ion of Groups I and II of the periodicchart, and of the formula:

wherein: X is of from 1 to 7 atoms other than hydrogen and is oxygen,sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;n is 0 or 1; R₁ is hydrogen or an organic group of from 1 to 12 carbonatoms and from 0 to 6 heteroatoms, which are chalcogen, nitrogen, andhalogen consisting of an aliphatic group of from 1 to 6 carbon atomshaving from 0 to 2 sites of unsaturation, non-oxo-carbonyl and thenitrogen and sulfur derivatives thereof, alicyclic having from 0 to 2sites of unsaturation, aryl, heterocyclic and combinations thereof,where the cyclic structures may have from 1 to 2 rings; R₂ is hydrogen,a heterofunctionality having nitrogen and/or chalcogen bonded to annularcarbon, a heterofunctionality having nitrogen and/or chalcogen bonded toannular carbon to which is substituted with an organic group of from 1to 10 carbon atoms, aryl, alkaryl, aralkyl and aralkenyl of from 5 to 10carbon atoms, aroyl of from 6 to 10 carbon atoms, or an organic groupbonded through a carbon atom of from 1 to 12 carbon atoms having from 1to 4, as described above for R₁; R₃ is hydrogen or an organic group offrom 1 to 10 carbon atoms and from 0 to 4 chalcogen and nitrogenheteroatoms; R₄ is hydrogen or alkyl and substituted alkyl of from 1 to6 carbon atoms, where the substituents are oxy, amino and halo; with theproviso that R₃ and R4 can be taken together to form a ring with theannular atoms to which they are attached of from 4 to 10 annular atomsand forming from 1 to 2 rings, where the annular atoms are unsubstitutedor substituted with halo, alkyl of from 1 to 3 carbon atoms, oxy of from0 to 3 carbon atoms, thio of from 0 to 3 carbon atoms and amino of from0 to 4 carbon atoms;

wherein: p is 0, 1 or 2; and R₅ is a group having from 1 to 3 atomsother than hydrogen and is oxy, thio, amino, nitro, cyano, and alkyl;

wherein: Y is O, S(O)_(m),, wherein m is 0, 1 or 2, amino or CH₂; R₆ isH or alkyl of from 1-3 carbon atoms; R₇ is hydrogen, or a group of from0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano,and alkyl; V is an aryl group having 6 annular members comprising 0 to 2nitrogen atoms and the remainder carbon atoms U is a substituent groupof from 0 to 5 atoms other than hydrogen, and is oxy, thio amino, nitro,cyano, halo, and alkyl; and u is 0 to 3; and (4)diazolohexahydroquinoline

wherein: Y is oxygen, sulfur, NH, (alkyl of from 1 to 3 carbon atoms, H)or H₂ R₇ is hydrogen or an organic group of from 1 to 12 carbon atomsand 0 to 4 heteroatoms; R₈ is hydrogen, an aliphatic group of from 1 to6 carbon atoms or a heterocycle of from 5 to 6 annular members and from1 to 2 heteroannular members that are oxygen, nitrogen or sulfur; andR₉, R₁₀, R₁₃, R₁₄, R₁₅ and R₁₆ are the same or different and arehydrogen or an organic radical of from 1 to 12 carbon atoms or aheterosusbtituent of from 1 to 3 heteroatoms; R₁₁ and R₁₂ are the sameor different and are hydrogen or an organic group of from 1 to 12 carbonatoms.
 2. A combination according to claim 1, wherein said polypeptideand said non-peptide organic molecule are complexed at the modulatingdomain of EPO-R.
 3. A combination according to claim 2, wherein saidpolypeptide is EPO-R bound to a cellular membrane.
 4. A combinationcomprising a polypeptide comprising the modulating domain sequence ofthe erythropoietin receptor and a non-peptide organic molecule of from12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, and from4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metal ionof Groups I and II of the periodic chart, and of the formula:

wherein: X is of from 1 to 3 atoms other than hydrogen and is oxygen,sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;n is 0 or 1; R₁ is a lower alkyl group of 1 to 3 carbon atoms or anorganic group having a six annular membered aromatic group having from 0to 3 substituents, where the substituents are halo, lower alkyl of from1 to 3 carbon atoms, nitro, trihalomethyl, and is either directly bondedto X or bonded through a linking group of from 1 to 4 carbon, nitrogen,or chalcogen atoms in the chain, being particularly carbon and nitrogen,and there being from 0 to 2 heteroatoms in the chain, where heteroatomsare bonded solely to carbon and hydrogen, or alpha-acetamidinyl havingfrom 0 to 1 N—OH; R₂ is hydrogen, amino of 0 to 3 carbon atoms, oxy offrom 0 to 3 carbon atoms, a heterofunctionality having nitrogen orchalcogen bonded to annular carbon to which is substituted an organicgroup of from 1 to 10 carbon atoms and from 0 to 3 heteroatoms; R₃ ishydrogen or an organic group of from 1 to 10 carbon atoms and from 0 to4 chalcogen and nitrogen heteroatoms; R₄ is hydrogen, alkyl orsubstituted alkyl of from 1 to 6 carbon atoms, where the substituentsare oxy, amino and halo; with the proviso that R₃ and R₄ can be takentogether to form a ring with the annular atoms to which they areattached of from 4 to 10 annular atoms and forming from 1 to 2 rings,where the annular atoms are unsubstituted or substituted with halo,alkyl of from 1 to 3 carbon atoms, oxy of from 0 to 3 carbon atoms, thioof from 0 to 3 carbon atoms and amino of from 0 to 4 carbon atoms.
 5. Acombination according to claim 4, wherein R₃ is hydrogen or an organicgroup of from 1 to 8 carbon atoms and 0 to 4 chalcogen, nitrogen andhalo heteroatoms.
 6. A combination according to claim 5, wherein R₃ iscyclopropylmethylamino.
 7. A combination according to claim 5, whereinR₃ is H.
 8. A combination according to claim 4, wherein R₁ is a sixannular membered aromatic group having from 0 to 3 substituents, wherethe substituents are halo, lower alkyl of from 1 to 3 carbon atoms,nitro, trihalomethyl, and is either directly bonded to X or bondedthrough a linking group of from 1 to 4 carbon, nitrogen, or chalcogenatoms in the chain.
 9. A combination according to claim 4, wherein R₄ ismethyl.
 10. A combination according to claim 4, wherein R₄ is H.
 11. Acombination comprising a polypeptide comprising the modulating sequenceof the erythropoietin receptor and a non-peptide organic molecule offrom 12 to 36 atoms other than hydrogen, from 9 to 20 carbon atoms, andfrom 4 to 12 of the heteroatoms chalcogen, nitrogen, halogen, and metalion of Groups I and II of the periodic chart, and of the formula:

wherein: Y is O, S(O)_(m),, wherein m is 0, 1 or 2, amino or CH₂; R₆ isH or alkyl of from 1-3 carbon atoms; R₇ is hydrogen, or a group of from0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro, cyano,and alkyl; V is a phenyl group; U is oxy, thio amino, nitro, cyano,halo, and alkyl and from 0 to 3 atoms other than hydrogen; and u is 0 to3.
 12. A combination according to claim 11, wherein Y is SO₂, V isphenyl, R₇ is Cl and u is
 0. 13. A combination comprising a polypeptidecomprising the modulating domain sequence of the erythropoietin receptorand a non-peptide organic molecule of from 12 to 36 atoms other thanhydrogen, from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatomschalcogen, nitrogen, halogen, and metal ion of Groups I and II of theperiodic chart, and of the formula:

wherein: X is of from 1 to 3 atoms other than hydrogen and is oxygen,sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino;n is 0 or 1; R₁ is alkyl of from 1 to 3 carbon atoms, substituted phenylhaving from 0 to 3 substituents that are CH₃, Cl, NO₂, and CF₃ andbonded directly to an annular carbon atom or through a linking group offrom 1 to 3 carbon and nitrogen atoms in the chain or N-hydroxyamidinyl;R₂ is CH₃, NH₂, OH, and aroylamido of from 7 to 8 carbon atoms havingfrom 0 to 2 susbtituents that are CH₃, Cl, NO₂, and CF₃; R₃ iscycloalkylalkyl of from 4 to 8 carbon atoms, having from 3 to 4 annularatoms, H or carboxy; R₄ is H, lower alkyl of from 1 to 3 carbon atoms oralkoxymethyl of from 2 to 4 carbon atoms; with the proviso that R₃ andR₄ may be taken together to define 1,2-dimethylene-alpha-halo,alpha-CH₃-halobenzene, where halo is F or Cl.
 14. A method formodulating the activity of EPO-R present as a cell membrane componentcomprising: forming a complex by bringing together the members of thecombination of claim 13 under complex forming conditions, where saidpolypeptide is EPO-R.
 15. A method for modulating the activity of EPO-Rcomprising: forming a complex by bringing together the members of thecombination of claim 11 under complex forming conditions, where saidpolypeptide is EPO-R.
 16. A compound according to claim 13 and apharmaceutically acceptable vehicle.
 17. A compound according to claim11 and a pharmaceutically acceptable vehicle.
 18. A method ofdetermining the binding affinity of a test compound to the modulatingdomain of EPO-R, said method comprising: adding said test compound to acombination according to claim 1 and determining the amount of complexof said combination in the presence of said test compound as compared tothe absence of said test compound.
 19. A method of inducing aphysiological response of EPO-R in a host, said method comprising:administering to said host a physiologically effective amount of anon-peptide organic molecule of from 12 to 36 atoms other than hydrogen,from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatomschalcogen, nitrogen, halogen, and metal ion of Groups I and II of theperiodic chart, and of the formula:

 wherein:  X is of from 1 to 3 atoms other than hydrogen and is oxygen,sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino; n is 0 or 1;  R₁ is alkyl of from 1 to 3 carbon atoms, substitutedphenyl having from 0 to 3 substituents that are CH₃, Cl, NO₂, and CF₃and bonded directly to an annular carbon atom or through a linking groupof from 1 to 3 carbon and nitrogen atoms in the chain,N-hydroxyamidinyl;  R₂ is CH₃, NH₂, OH, and aroylamido of from 7 to 8carbon atoms having from 0 to 2 susbtituents that are CH₃, Cl, NO₂, andCF₃;  R₃ is cycloalkylalkyl of from 4 to 8 carbon atoms, having from 3to 4 annular atoms, H or carboxy;  R₄ is H, lower alkyl of from 1 to 3carbon atoms or alkoxymethyl of from 2 to 4 carbon atoms;  with theproviso that R₃ and R₄ may be taken together to define1,2-dimethylene-alpha-halo, alpha-CH₃-halobenzene, where halo is F orCl; or

 wherein:  X is of from 1 to 3 atoms other than hydrogen and is oxygen,sulfur bonded to 0 to 2 oxygen atoms, amino and alkyl substituted amino; n is 0 or 1;  Y is O, S(O)_(m),, wherein m is 0, 1 or 2, amino or CH₂; R₆ is H or alkyl of from 1-3 carbon atoms;  R₇ is hydrogen, or a groupof from 0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro,cyano, and alkyl;  V is a phenyl group;  U is oxy, thio amino, nitro,cyano, halo, and alkyl and from 0 to 3 atoms other than hydrogen; and  uis 0 to
 3. 20. A method according to claim 19, wherein said non-peptideorganic molecule is of formula
 1. 21. A method according to claim 20,wherein X is amino, R₂ is o-methyl, p-chlorophenyl-1, R₂ is H, R₃ iscyclopropylmethylamino and R₄ is methyl.
 22. A method of modulating theresponse to a stimulus of hematopoietic or neuronal cells influenced bythe binding of EPO to EPO-R, said method comprising: contacting saidcells with an effective amount to modulate said response of anon-peptide organic molecule of from 12 to 36 atoms other than hydrogen,from 9 to 20 carbon atoms, and from 4 to 12 of the heteroatomschalcogen, nitrogen, halogen, and metal ion of Groups I and II of theperiodic chart, and of the formula:

 wherein:  R₁ is alkyl of from 1 to 3 carbon atoms, substituted phenylhaving from 0 to 3 substituents that are CH₃, Cl, NO₂, and CF₃ andbonded directly to an annular carbon atom or through a linking group offrom 1 to 3 carbon and nitrogen atoms in the chain, N-hydroxyamidinyl; R₂ is CH₃, NH₂, OH, and aroylamido of from 7 to 8 carbon atoms havingfrom 0 to 2 susbtituents that are CH₃, Cl, NO₂, and CF₃;  R₃ iscycloalkylalkyl of from 4 to 8 carbon atoms, having from 3 to 4 annularatoms, H or carboxy;  R₄ is H, lower alkyl of from 1 to 3 carbon atomsor alkoxymethyl of from 2 to 4 carbon atoms;  with the proviso that R₃and R₄ may be taken together to define 1,2-dimethylene-alpha-halo,alpha-CH₃-halobenzene, where halo is F or Cl; or

 wherein:  Y is O, S(O)_(m),, wherein m is 0, 1 or 2, amino or CH₂;  R₆is H or alkyl of from 1-3 carbon atoms;  R₇ is hydrogen, or a group offrom 0 to 3 atoms other than hydrogen, and is oxy, thio amino, nitro,cyano, and alkyl;  V is a phenyl group;  U is oxy, thio amino, nitro,cyano, halo, and alkyl and from 0 to 3 atoms other than hydrogen; and  uis 0 to 3.